EP4370136A2 - Einfache chemische ansätze zum einbringen von 2,6-diaminopurin- und 2-aminoadeninkonjugaten in oligonukleotide - Google Patents

Einfache chemische ansätze zum einbringen von 2,6-diaminopurin- und 2-aminoadeninkonjugaten in oligonukleotide

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Publication number
EP4370136A2
EP4370136A2 EP22842917.1A EP22842917A EP4370136A2 EP 4370136 A2 EP4370136 A2 EP 4370136A2 EP 22842917 A EP22842917 A EP 22842917A EP 4370136 A2 EP4370136 A2 EP 4370136A2
Authority
EP
European Patent Office
Prior art keywords
optionally substituted
alkyl
group
nucleotide
oligonucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22842917.1A
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English (en)
French (fr)
Inventor
Muthiah Manoharan
Mimouna MADAOUI
Dhrubajyoti Datta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alnylam Pharmaceuticals Inc
Original Assignee
Alnylam Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Alnylam Pharmaceuticals Inc filed Critical Alnylam Pharmaceuticals Inc
Publication of EP4370136A2 publication Critical patent/EP4370136A2/de
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical

Definitions

  • the present disclosure relates generally to methods and compositions for facile synthesis of oligonucleotide comprising 2,6-diaminopurine and 2-aminoadenine conjugates.
  • the 2,6-diaminopurine (DAP) nucleobase can form Watson-Crick base pairs with thymine in DNA and uracil in RNA. These pairs are stabilized by three hydrogen bonds resulting in improved thermodynamic duplex stability relative to base pairing with adenine.
  • Previously reported strategies for synthesis of DAP building blocks for oligonucleotide synthesis require multiple steps and have -NEU protecting group issues.
  • the inventors have developed a post-synthetic strategy using the 2-fluoro-6-amino- adenine as the key intermediate to make DAP-containing oligonucleotides and their conjugates.
  • oligonucleotide comprising a nucleoside of Formula (I):
  • the oligonucleotide comprising a nucleotide of Formula II can be linked to a solid support when reacting with an amine of formula HNR 6 R 7 .
  • the oligonucleotide comprising a nucleotide of Formula II is not linked to a solid support when reacting with an amine of formula HNR 6 R 7 .
  • the N atom in the amine of formula HNR 6 R 7 can be 15 N.
  • oligonucleotide comprising a nucleoside of Formula (X):
  • the method comprises reacting an oligonucleotide comprising a nucleoside of Formula
  • alkali hydroxide or alkali earth hydroxide e.g., NaOH
  • the oligonucleotide comprising a nucleotide of Formula XI can be linked to a solid support when reacting with an alkali hydroxide or alkali earth hydroxide (e.g., NaOH).
  • an alkali hydroxide or alkali earth hydroxide e.g., NaOH
  • the oligonucleotide comprising a nucleotide of Formula XI is not linked to a solid support when reacting with an alkali hydroxide or alkali earth hydroxide (e.g., NaOH).
  • the oligonucleotide comprising a nucleotide of Formula XI is linked to a solid support and the method comprises a step of cleaving the oligonucleotide from the solid support prior to reacting with an alkali hydroxide or alkali earth hydroxide (e.g., NaOH).
  • an alkali hydroxide or alkali earth hydroxide e.g., NaOH.
  • R H is halogen (e.g., chloro or fluoro);
  • R 2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4-30 alkyl- 0N(CH 2 R 8 )(CH 2 R 9 ), a bond to an intemucleotide linkage to a subsequent nucleotide, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide
  • R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, hydrogen, hydroxy, protected hydroxy, optionally substituted C 1-30 alkyl, optionally substituted C 2 - 3 0 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl- 0N(CH 2 R 8 )(CH 2 R 9 ), a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a ligand, a linker covalently bonded to
  • R 4 is hydrogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, or optionally substituted C 1-6 alkoxy; or R 4 and R 2 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4 , -Y-C(R 10 R 11 ) v -2’;
  • Y is -O-, -CH 2 -, -CH(Me)-, -C(CH 3 ) 2 -, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, - S(O) 2 -, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 )-;
  • R 10 and R 11 independently are H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl or optionally substituted C 2 -C 6 alkynyl;
  • R 12 is hydrogen, optionally substituted C 1-30 alkyl, optionally substituted C 1 - C 30 alkoxy, C 1-4 haloalkyl, optionally substituted C 2-4 alkenyl, optionally substituted C 2-4 alkynyl, optionally substituted C 1-30 alkyl-CO 2 H, or a nitrogen-protecting group;
  • v is 1, 2 or 3; or R 4 and R 3 taken together with the atoms to which they are attached form an optionally substituted C 3-8 cycloalkyl, optionally substituted C 3-8 cycloalkenyl, or optionally substituted 3-8 membered heterocyclyl;
  • R 5 represents a bond to an intemucleotide linkage to a preceding nucleotide, hydrogen, hydroxy, protected hydroxy, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, optionally substituted 3-8 membered heterocyclyl (e.g., morpholin-l-yl, piperidin-1- yl, orpyrrolidin-l-yl), halogen, alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4 - 3 0 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group, C
  • each R L is absent or a linker; each R L is a ligand, (e.g., selected independently from the group consisting of carbohydrates, peptides, lipids, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, vitamins, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl); each R 8 and R 9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, provided that,
  • a targeting ligand e.g., GalNac
  • a pharmacokinetics modifier optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, provided that,
  • R 5 is a bond to an intemucleotide linkage to a preceding nucleotide.
  • R H is halogen
  • R 32 is hydrogen, hydroxy, halogen protected hydroxy, phosphate group, reactive phosphorous group , optionally substituted C 1-30 alkyl, optionally substituted C 2 - 3 0 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy), , alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl- 0N(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support;
  • R 33 is hydrogen, hydroxy, halogen protected hydroxy, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 2 - 3 0 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy), , alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl- 0N(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support, and optionally, only one of R 32 and R 33 is a
  • R 4 is hydrogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkynyl, or optionally substituted C 1-6 alkoxy; or R 4 and R 32 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y-C(R 10 R 11 ) v -2’;
  • Y is -O-, -CH 2 -, -CH(Me)-, -C(CH 3 ) 2 -, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, - S(O) 2 -, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 )-;
  • R 10 and R 11 independently are H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl or optionally substituted C 2 -C 6 alkynyl;
  • R 12 is hydrogen, optionally substituted C 1-30 alkyl, optionally substituted C 1 - C 30 alkoxy, C 1-4 haloalkyl, optionally substituted C 2-4 alkenyl, optionally substituted C 2-4 alkynyl, optionally substituted C 1-30 alky-CO 2 H, or a nitrogen-protecting group; v is 1, 2 or 3; or R 4 and R 33 taken together with the atoms to which they are attached form an optionally substituted C 3-8 cycloalkyl, optionally substituted C 3-8 cycloalkenyl, or optionally substituted 3-8 membered heterocyclyl;
  • R 35 is hydroxy, protected hydroxy, phosphate group, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4 - 3 0 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group, C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate), monophosphate ((HO) 2 (O)P-O-5'), diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'), triphosphat
  • R 35 is a vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic; and R 33 is a reactive phosphorous group.
  • VP vinylphosphonate
  • R 35 is a vinylphosphonate (VP) group, cyclopropylphosphonate, or a phosphate mimic; and R 33 is a reactive phosphorous group.
  • VP vinylphosphonate
  • R 33 is a reactive phosphorous group.
  • R 35 is a vinylphosphonate (VP) group (e.g., E- vinylphosphonate), cyclopropylphosphonate, or a phosphate mimic; and R 33 is a phosphoramidite group.
  • VP vinylphosphonate
  • R 35 is a triphosphate group and R 33 is allyloxy, azidomethoxy, or aminooxy.
  • R 33 is a reactive phosphorous group.
  • R 35 is a vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic; and R 33 is a reactive phosphorous group.
  • VP vinylphosphonate
  • R 35 is a vinylphosphonate (VP) group, cyclopropylphosphonate, or a phosphate mimic; and R 33 is a reactive phosphorous group.
  • R 35 is a vinylphosphonate (VP) group (e.g., E- vinylphosphonate), cyclopropylphosphonate, or a phosphate mimic; and R 33 is a phosphoramidite group.
  • R 35 is a triphosphate group and R 33 is allyloxy, azidomethoxy, or aminooxy.
  • R 33 is a reactive phosphorous group.
  • Some exemplary compounds of Formula (III) include, but are not limited to the following:
  • Additional exemplary compounds of Formula (III) include, but are not limited to,
  • an oligonucleotide prepared using a method described herein.
  • the disclosure provides an oligonucleotide comprising nucleoside of Formula (I), (II), (X) and/or (XI).
  • the nucleoside of Formula (I), (II), (X) or (XI) can be present in any position in the oligonucleotide.
  • a nucleoside of Formula (I), (II), (X) or (XI) can be at the 5 ’-end of the oligonucleotide.
  • a nucleoside of Formula (I), (II), (X) or (XI) can be at the 3 ’-end of the oligonucleotide. In yet some other non-limiting examples, a nucleoside of Formula (I), (II), (X) or (XI) can be present at an internal position of the oligonucleotide.
  • FIG. 1 is a schematic representation of exemplary aspects of 2,6-diaminopurine (DAP) nucleobase.
  • FIG. 2 shows exemplary 2-fluoro-6-aminopurine monomers for oligonucleotide synthesis.
  • FIG. 3 shows exemplary monomer residues after incorporation into oligonucleotides.
  • FIGS. 4 and 5 are schematic representation of post-synthesis conjugation according to exemplary embodiments of the invention.
  • FIG. 6 shows exemplary ligands the could be conjugated at the 2-amino of the DAP.
  • FIGS. 7A and 7B show exemplary post-synthesis conjugation monomer residues in oligonucleotides according to some exemplary embodiments of the disclosure.
  • FIG. 8 show exemplary post-synthesis conjugation monomer residues in oligonucleotides according to some exemplary embodiments of the disclosure.
  • FIG. 9 is a synthetic scheme for synthesis of 2-fluoro-6-aminopurine nucleosides.
  • References for compounds 1, 3, 5, 7, 8 and 9 are Morris et. al. J Med. Chem. 1972, 15, 735; Vorbrueggen et. al. Nucleosides & Nucleotides 1994, 13, 673; Iribarren et. al. Nucleic Acids Research 1990, 18, 41; Ross et. al. Nucleosides, Nucleotides & Nucleic Acids 2008, 27, 67; Koch et. al. BioorgMed Chem 2004, 12, 2385; Ross & Manoharan. 2000, W02000012563; Manoharan et. al. in PCT Int. Appl. 2009, WO 2009091982; and Montgomery et. al. Nucleosides & Nucleotides 1994, 13, 309.
  • FIG. 10 is a schematic representation of synthesis of exemplary oligonucleotide comprising DAP nucleoside.
  • FIG. 11 shows ion-exchange HPLC analysis of crude and purified oligonucleotides after site-specific single DAP incorporation.
  • FIG. 12 shows LCMS analysis of purified oligonucleotides after site-specific single DAP incorporation.
  • FIG. 13 is a schematic representation of post-synthetic conjugation according to an embodiment of the invention.
  • FIGS. 14-16 shows LCMS analysis of crude oligonucleotides after post-synthetic conjugation on solid support (FIGS. 14 and 15) and solution phase (FIG. 16).
  • FIG. 17 is a schematic representation of some exemplary DAP conjugates.
  • FIG. 18 shows 19 F NMR analysis of any oligonucleotide prepared according to an exemplary embodiment of the method described herein.
  • FIG. 19 is a schematic representation showing conversion of 2-F A to iso- G according to an exemplary embodiment of the method described herein.
  • FIG. 20 shows LC-MS analysis of iso-G comprising oligonucleotides prepared according to an exemplary embodiment of the method described herein.
  • oligonucleotide comprising a nucleoside of Formula (X):
  • the method comprises reacting an oligonucleotide comprising a nucleoside of Formula (XI): with an alkali hydroxide or alkali earth hydroxide (e.g., NaOH).
  • an alkali hydroxide or alkali earth hydroxide e.g., NaOH
  • R H is halogen.
  • R H can be fluoro, chloro, bromo or iodo. In some embodiments of any one of the aspects described herein, R H is fluoro.
  • each R L can be independently selected from the groups consisting of H, carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucelsides and nucleotides, oligonucleotides, detectable labels, diagnostic agents (e.g., bitoin), fluorescent dyes, polyethylene glycols (PEGs), antibodies, antibody fragments (e.g., nanobodies).
  • diagnostic agents e.g., bitoin
  • fluorescent dyes e.g., polyethylene glycols (PEGs)
  • PEGs polyethylene glycols
  • antibodies e.g., nanobodies
  • R L is a ligand.
  • ligands modify one or more properties of the attached molecule (e.g., the oligonucleotide described herein) including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Ligands are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound.
  • a preferred list of ligands includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • Preferred ligands amenable to the present invention include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thi
  • Ligands can include naturally occurring molecules, or recombinant or synthetic molecules.
  • exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2 , polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine
  • porphyrins e.g., TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g., EDTA
  • lipophilic molecules e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1- pyrene butyric acid, dihydrotestosterone, l,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytr
  • biotin transport/absorption facilitators
  • transport/absorption facilitators e.g., naproxen, aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactors, lipopolysaccharide, an activator of p38 MAP kinase, an activator of NF-KB, taxon, vincristine, vinblastine, cytochalasin, nocodazole,
  • Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, b, or g peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
  • a peptidomimetic also referred to herein as an oligopeptidomimetic is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide.
  • the peptide or peptidomimetic ligand can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • amphipathic peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H 2 A peptides, Xenopus peptides, esculentinis-1, and caerins.
  • endosomolytic ligand refers to molecules having endosomolytic properties.
  • Endosomolytic ligands promote the lysis of and/or transport of the composition of the invention, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell.
  • Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and brached polyamines, e.g.
  • spermine cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.
  • Exemplary endosomolytic/fusogenic peptides include, but are not limited to, AALEALAEALEALAEALEALAEAAAAGGC (GALA);
  • AALAEALAEALAEALAEALAEALAAAAGGC (EALA); ALEALAEALEALAEA; GLFEAIEGFIENGWEGMIWDYG (INF-7); GLF GAI AGFIENGWEGMIDGWY G (Inf HA-2); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3);
  • GLF GLF G ALAEALAEALAEHL AEALAEALE ALA AGG SC (GLF);
  • GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, n is norleucine); LFEALLELLESLWELLLEA (JTS-1); GLFKALLKLLKSLWKLLLKA (ppTGl); GLFRALLRLLRSLWRLLLRA (ppTG20); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA); GLFFEAIAEFIEGGWEGLIEGC (HA); GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin); H 5 WYG; and CHK 6 HC.
  • fusogenic lipids fuse with and consequently destabilize a membrane.
  • Fusogenic lipids usually have small head groups and unsaturated acyl chains.
  • Exemplary fusogenic lipids include, but are not limited to, l,2-dileoyl-sn-3- phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen- 19-ol (Di-Lin), N-methyl(2,2-di((9Z, 12Z)-octadeca-9, 12-dienyl)- 1 ,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z, 12Z)-octadeca-9,
  • Exemplary cell permeation peptides include, but are not limited to,
  • LLIILRRRIRKQ AH AH SK PVEC
  • GWTLN S AGYLLKINLKALAALAKKIL transportan
  • KLALKLALKALKAALKLA amphiphilic model peptide
  • RRRRRRRRR Arg9
  • KFFKFFKFFK Bacillus FFK
  • LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNILVPRTES LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNILVPRTES (LL-37); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin PI);
  • ACY CRIP ACI AGERRY GTCIY QGRLWAFCC (a-defensin); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (p-defensin); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39); ILPWKWPWWPWRR-NH2 (indolicidin); AAVALLPAVLLALLAP (RFGF); AALLPVLLAAP (RFGF analogue); and RKCRIWIRVCR (bactenecin).
  • NH 2 alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid
  • NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).
  • targeting ligand refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment.
  • Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.
  • Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacids and lectins.
  • the term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.
  • PK modulating ligand and “PK modulator” refers to molecules which can modulate the pharmacokinetics of oligonucleotides described herein.
  • Some exemplary PK modulator include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, and transthyretia-binding ligands (e.g., tetraiidothyroacetic acid, 2, 4, 6-triiodophenol and flufenamic acid).
  • Oligomeric compounds that comprise a number of phosphorothioate intersugar linkages are also known to bind to serum protein, thus short oligomeric compounds, e.g. oligonucleotides of comprising from about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), and that comprise a plurality of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).
  • ligands e.g. as PK modulating ligands
  • the PK modulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and/or phosphorodithioate linkages. In some embodiments, all intemucleoside linkages in PK modulating oligonucleotide are phosphorothioate and/or phosphorodithioates linkages.
  • aptamers that bind serum components e.g. serum proteins
  • Binding to serum components can be predicted from albumin binding assays, scuh as those described in Oravcova, et al., Journal of Chromatography B (1996), 677: 1-27.
  • the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties.
  • a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties.
  • all the ligands have different properties.
  • the ligand has a structure shown in any of Formula (IV) - (VII): wherein: q 2A q 2B , q 3A q 3B q4 A q 4B q 5A q 5B and q 5C represent independently for each occurrence
  • repeating unit can be the same or different
  • P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , T 5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O;
  • L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represent the ligand; i.e.
  • a monosaccharide such as GalNAc
  • disaccharide such as GalNAc
  • trisaccharide such as glycerol
  • tetrasaccharide such as oligosaccharide
  • polysaccharide such as glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, a saccharide, oligosaccharide, or polysaccharide
  • R a is H or amino acid side chain.
  • the ligand is of Formula (VII): wherein L 5A , L 5B and L 5C represent a monosaccharide, such as GalNAc derivative.
  • Exemplary ligands include, but are not limited to, the following: [0019] In some embodiments of any one of the aspects described herein, the ligand is a ligand described in US Patent No. 5,994,517 or US Patent No. 6,906,182, content of each of which is incorporated herein by reference in its entirety.
  • the ligand can be a tri-antennary ligand described in Figure 3 of US Patent No. 6,906,182.
  • the ligand is selected from the following tri-antennary ligands: [0021]
  • R L is a ligand.
  • R L when more than one R L are present, they can be same or different. Accordingly, in some embodiments of any one of the aspects described herein, all R L are same. In some other embodiments of any one of the aspects described herein, R L are different.
  • R 7 is not H.
  • at least one of R 6 and R 7 is -L-R l .
  • R 6 and R 7 can be same or different. Accordingly, in some embodiments of any one of the aspects described herein, R 6 and R 7 are the same. In some other embodiments of any one of the aspects described herein, R 6 and R 7 are different.
  • one of R 6 and R 7 is H.
  • R 7 is selected independently from the group consisting of: cyclo [Phe-Arg-Gly-Asp-Leu-Ala-Phe-D-Pro-N-Me-Lys(PEG4-NH2)] (cyclic RGD peptide) or optionally substituted C 1 -C 30 alkyl, and wherein R is
  • At least one of R 6 and R 7 is an optionally substituted, branched C 4 -C 30 alkyl.
  • at least one of R 6 and R 7 is -CHR L1 R L2 , where R L1 and R L2 are independently selected from optionally substituted C 1 -C 15 alkyls.
  • R L1 is optionally substituted C 6 -C 12 alkyl, e.g., C 7 , C 8 , C 9 , C 10 , or C 11 alkyl, each of which can be optionally substituted. In some embodiments, R L1 is optionally substituted C 8 alkyl.
  • R L2 is is optionally substituted C 6 -C 12 alkyl, e.g., C 7 , C 8 , C 9 , C 10 , or C 11 alkyl, each of which can be optionally substituted. In some embodiments, R L2 is optionally substituted C 7 alkyl.
  • R 6 and R 7 independently are optionally substituted C 1 -C 30 alkyl.
  • R 6 and R 7 independently are optionally substituted C 4 -C 20 alkyl, e.g., R 6 and R 7 independently are optionally substituted C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , 5 12 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 alkyl.
  • At least one of R 6 and R 7 is -CH 2 (CH 2 )MCO 2 H.
  • R 2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2 - 30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O- C 4 - 3 oalkyl-ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4 - 3 oalkyl-ON(CH 2 R 8 )(CH 2 R 9 ).
  • alkoxyalkyl e.g., methoxyethyl
  • alkoxyalkylamine alkoxycarboxylate
  • R 2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.
  • alkoxyalkyl e.g., methoxyethyl
  • alkoxyalkylamine e.g., methoxyethyl
  • alkoxycarboxylate amino, alkylamino, or dialkylamino.
  • R 2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkoxy, or alkoxyalkyl (e.g., methoxyethyl. In some embodiments of any one of the aspects, R 2 is hydrogen, hydroxy, protected hydroxy, fluoro or methoxy.
  • R 2 is halogen.
  • R 2 can be fluoro, chloro, bromo or iodo. In some embodiments of any one of the aspects described herein, R 2 is fluoro.
  • R 2 and R 4 [0036] In some embodiments of any one of the aspects described herein, R 2 and R 4 taken together are d’-C(R1VY-2’ or 4’-Y-C(R 10 R 11 ) v -2’; v is 1, 2 or 3; where Y is -O-, -CH 2 -, - CH(Me)-, -C(CH 3 ) 2 -, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, - N(R 12 )C(O)-, or -C(O)N(R 12 )-; R 10 and R 11 independently are H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 al
  • v is 1. In some other embodiments of any one of the aspects, v is 2.
  • Y is O.
  • R 2 and R 4 taken together are 4’- C(R 10 R 11 ) v -O-2’.
  • R 10 and R 11 attached to the same carbon can be same or different.
  • one of R 10 and R 11 can be H and the other of the R 10 and R 11 can be an optionally substituted C 1 -C 6 alkyl.
  • R 10 and R 11 independently are H or C 1 -C 30 aIkyI optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 aIkoxy.
  • one of R 10 and R 11 is H and the other is C 1 -C 6 alkyl, optionally substituted with a C 1 -C 6 alkoxy.
  • one of R 10 and R 11 is H and the other is -CH 3 or CH 2 OCH 3 .
  • R 10 and R 11 attached to the same C are the same.
  • R 10 and R 11 attached to the same C are H.
  • R 2 and R 4 taken together are 4’-CH 2 - O-2’, 4’-CH(CH 3 )-O-2’, 4’-CH(CH 2 OCH 3 )-O-2’, or 4’- CH 2 CH 2 -O-2’.
  • R 2 and R 4 taken together are 4’- CH 2 CH 2 -O-2’.
  • R 2 is a bond to an intemucleotide linkage to a subsequent nucleotide. It is noted that only one of R 2 and R 3 can be a bond to an intemucleotide linkage to a subsequent nucleotide.
  • R 3 can be a bond to an intemucleotide linkage to a subsequent nucleotide, hydroxy, protected hydroxy, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), amino, alkylamino, dialkylamino, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a ligand, a linker covalently bonded to one or more ligands (e.g., N- acetylgalactosamine (GalNac)), a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • alkoxyalkyl e.g., methoxyethyl
  • amino alkylamino, dialkylamino
  • R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, hydroxy, protected hydroxy, optionally substituted C 1-30 alkoxy, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • a 3’-oligonuclotide capping group e.g., an inverted nucleotide or an inverted abasic nucleotide
  • a linker covalently bonded e.g., - C(O)CH 2 CH 2 C(O)-
  • R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, hydroxy, a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide.
  • R 3 is a solid support, or a linker covalently bonded to a solid support.
  • R 3 is hydroxyl.
  • R 3 and R 4 taken together with the atoms to which they are attached form an optionally substituted C 3-8 cycloalkyl, optionally substituted C 3-8 cycloalkenyl, or optionally substituted 3-8 membered heterocyclyl.
  • R 4 can be hydrogen, optionally substituted C 1-6 alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2 - 6 alkynyl, or optionally substituted C 1-6 alkoxy.
  • R 4 can be hydrogen, optionally substituted C 1-6 alkyl or optionally substituted C 1-6 alkoxy.
  • R 4 is H.
  • R 5 can be a bond to an intemucleotide linkage to a preceding nucleotide, hydrogen, hydroxy, protected hydroxy, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2 - 30 alkynyl, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl- 0N(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group, monophosphate ((HO) 2 (O)P-O-5'), diphosphate ((HO) 2 (O)P-O-
  • R 5 can be a bond to an intemucleotide linkage to a preceding nucleotide, hydroxy, protected hydroxy, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate (phosphorodithioate), phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, or alkylphosphonates .
  • VP vinylphosphonate
  • R 5 is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxy, protected hydroxy, optionally substituted C 2-30 alkenyl, optionally substituted C 1-30 alkoxy or a vinylphosphonate (VP) group.
  • R 5 is a bond to an intemucleotide linkage to a preceding nucleotide.
  • R 5 is a hydroxyl or protected hydroxyl.
  • R 5 is optionally substituted C 2-30 alkenyl or optionally substituted C 1-30 alkoxy.
  • R 5 is a vinylphosphonate group.
  • R 5 can be -CH(R 51 )- X 5 -R 52 , where X 5 is absent, a bond or O; R 51 is hydrogen, optionally substituted C 1-30 alkyl, optionally substituted -C 2-30 alkenyl, or optionally substituted -C 2-30 alkynyl, and R 52 is a bond to an intemucleoside linkage to the preceding nucleotide.
  • X 5 is O or a bond.
  • X 5 is O.
  • X 5 is absent, i.e., R 5 is-CH(R 51 )R 52 .
  • R 5 is -CH(R 51 )-X 5 -R 52 .
  • R 51 is H.
  • R 51 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 - C 6 alkoxy.
  • R 51 is H.
  • R 51 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 - C 6 alkoxy.
  • R 52 is a bond to an intemucleoside linkage to the preceding nucleotide.
  • R 5 is optionally substituted C 1- 6 alkyl-R 53 , optionally substituted -C 2-6 alkenyl-R 53 , or optionally substituted -C 2-6 alkynyl-R 53 .
  • R 53 can be -OR 54 , -SR 55 , -P(O)(OR 56 ) 2 , - P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , -OP(O)(OR 56 ) 2 , -OP(S)(OR 56 ) 2 ,
  • R 54 is hydrogen or oxygen protecting group
  • R 55 is hydrogen or sulfur protecting group
  • each R 56 is independently hydrogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl, or an oxygen-protecting group
  • each R 57 is independently hydrogen, optionally substituted C 1-30 alkyl, optionally substituted C 2 - 30 alkenyl, or optionally substituted C 2-30 alkynyl, or a sulfur-protecting group.
  • SP(O)(OR 56 ) 2 , -SP(S)(OR 56 ) 2 , and -SP(S)(SR 57 )(OR 56 ) is hydrogen.
  • SP(O)(OR 56 ) 2 , -SP(S)(OR 56 ) 2 , or -SP(S)(SR 57 )(OR 56 ) is not hydrogen.
  • at least one at least one R 56 in P(O)(OR 56 ) 2 , -P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 ) 2 , -OP(S)(OR 56 ) 2 , - OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 ) 2 , -SP(S)(OR 56 ) 2 , and -SP(S)(SR 57 )(OR 56 ) is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl, or an oxygen-protecting group.
  • At least one R 56 is H and at least one R 56 is other than H in -P(O)(OR 56 ) 2 , -P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 ) 2 , - OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), SP(O)(OR 56 ) 2 , -SP(S)(OR 56 ) 2 , and -SP(S)(SR 57 )(OR 56 ).
  • all R 56 are H in -P(O)(OR 56 ) 2 , - P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 ) 2 , -OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), -
  • all R 56 are other than H in in - P(O)(OR 56 ) 2 , -P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -OP(O)(OR 56 ) 2 , -OP(S)(OR 56 ) 2 , -
  • At least one R 57 in -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , -OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 ) 2 , -SP(S)(SR 57 )(OR 56 ), and - SP(S)(SR 57 ) 2 is H.
  • At least one R 57 in -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , -OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 ) 2 , -SP(S)(SR 57 )(OR 56 ), and - SP(S)(SR 57 ) 2 is other than H.
  • At least one R 57 in -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , - OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 ) 2 , -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 ) 2 is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2 - 30 alkynyl, or an sulfur-protecting group.
  • At least one R 57 is H and at least one R 57 is other than H in -P(S)(SR 57 ) 2 , -OP(S)(SR 57 ) 2 and -SP(S)(SR 57 ) 2 .
  • all R 57 are H in -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , -OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 ) 2 , -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 ) 2 .
  • all R 57 are other than H in -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , - OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 ) 2 , -SP(S)(SR 57 )(OR 56 ), and -SP(S)(SR 57 ) 2 .
  • R 5 is optionally substituted -C 2-6 alkenyl-R 53 .
  • R 54 is hydrogen or an oxygen protecting group.
  • R 54 is hydrogen or 4,4'-dimethoxytrityl (DMT).
  • DMT 4,4'-dimethoxytrityl
  • R 54 is H.
  • R 5 is optionally substituted -C 1-6 alkenyl-R 53 .
  • R 5 can be -CH(R 58 )- R 53 , where R 53 is -OR 54 , -SR 55 , -P(O)(OR 56 ) 2 , -P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , - OP(O)(OR 56 ) 2 , -OP(S)(OR 56 ) 2 , -OP(S)(SR 57 )(OR 56 ), -OP(S)(SR 57 ) 2 , -SP(O)(OR 56 ) 2 , -
  • R 58 is H, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl.
  • R 58 is H. In some other non-limiting examples, R 58 is C 1 -C 30 alkyl optionally substituted with a substituent selected from NH 2 , OH, C(O)NH 2 , COOH, halo, SH, and C 1 -C 6 alkoxy.
  • R 5 is -CH(R 58 )-O- R 59 , where R 59 is H, -P(O)(OR 56 ) 2 , -P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , -OP(O)(OR 56 ) 2 .
  • R 5 is -CH(R 58 )-O-R 59 , where R 58 is H or optionally substituted C 1 -C 30 alkyl and R 59 is H or -P(O)(OR 56 ) 2 .
  • R 5 is -CH(R 58 )-S- R 60 , where R 60 is H, -P(O)(OR 56 ) 2 , -P(S)(OR 56 ) 2 , -P(S)(SR 57 )(OR 56 ), -P(S)(SR 57 ) 2 , -OP(O)(OR 56 ) 2 .
  • R 32 is hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyI, C 1-30 haloalkyl, optionally substituted C 2 - 30 alkenyI, optionally substituted C 2-30 alkynyI, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 325 , NHC(O)R 326 , a lipid, a link
  • R 322 can be H, hydroxyl protecting group, optionally substituted C 1-30 alkyI, C 1- 30 haloalkyl, optionally substituted C 2-30 alkenyI, optionally substituted C 2-30 alkynyI, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 323 can be H, sulfur protecting group, optionally substituted C 1-30 alkyI, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyI, optionally substituted C 2-30 alkynyI, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 324 can be H, hydroxyl protecting group, optionally substituted C 1-30 aIkyI, C 1- 30 haloalkyl, optionally substituted C 2-30 alkenyI, optionally substituted C 2-30 alkynyI, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 325 can be hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1-30 alkyI, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyI, optionally substituted C 2-30 alkynyI, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.
  • R 326 can be can be hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1- 3 oalkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.
  • R 32 is R 32 is hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2 - 30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 325 , NHC(O)R 324 .
  • R 32 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2 - 30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O- C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ).
  • R 2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.
  • alkoxyalkyl e.g., methoxyethyl
  • alkoxyalkylamine e.g., methoxyethyl
  • alkoxycarboxylate amino, alkylamino, or dialkylamino.
  • R 32 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkoxy, or alkoxyalkyl (e.g., methoxyethyl.
  • R 2 is hydrogen, hydroxy, protected hydroxy, fluoro or methoxy.
  • R 32 is halogen.
  • R 32 can be fluoro, chloro, bromo or iodo. In some embodiments of any one of the aspects described herein, R 32 is fluoro.
  • R 32 and R 4 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y-C(R 10 R 11 ) v -2’; v is 1, 2 or 3; where Y is -O-, -CH 2 -, - CH(Me)-, -C(CH 3 ) 2 -, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, - N(R 12 )C(O)-, or -C(O)N(R 12 )-; R 10 and R 11 independently are H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl or optionally substituted C 2 -C 6 alkyn
  • v is 1. In some other embodiments of any one of the aspects, v is 2. In some embodiments, Y is O.
  • R 32 and R 4 taken together are 4’-C(R 10 R 11 ) v -O-2’.
  • R 10 and R 11 attached to the same carbon can be same or different.
  • one of R 10 and R 11 can be H and the other of the R 10 and R 11 can be an optionally substituted C 1 -C 6 alkyl.
  • R 10 and R 11 independently are H or C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • one of R 10 and R 11 is H and the other is C 1 -C 6 alkyl, optionally substituted with a C 1 -C 6 alkoxy.
  • one of R 10 and R 11 is H and the other is -CH 3 or CH 2 OCH 3 .
  • R 10 and R 11 attached to the same C are the same.
  • R 10 and R 11 attached to the same C are H.
  • R 32 and R 4 taken together are 4’-CH 2 - O-2’, 4’-CH(CH 3 )-O-2’, 4’-CH(CH 2 OCH 3 )-O-2’, or 4’- CH 2 CH 2 -O-2’.
  • R 32 and R 4 taken together are 4’- CH 2 CH 2 -O-2’.
  • R 32 is a reactive phosphorus group.
  • reactive phosphorus groups are useful for forming intemucleoside linkages including for example phosphodiester and phosphorothioate intemucleoside linkages.
  • Such reactive phosphorus groups are known in the art and contain phosphorus atoms in P III or P V valence state including, but not limited to, phosphoramidite, H- phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries.
  • Reactive phosphorous group in the form of phosphoramidites (P III chemistry) as reactive phosphites are a preferred reactive phosphorous group for solid phase oligonucleotide synthesis.
  • the intermediate phosphite compounds are subsequently oxidized to the Pv state using known methods to yield phosphodiester or phosphorothioate intemucleoside linkages.
  • the reactive phosphorous group is -OP(OR P )(N(R P2 ) 2 ), -OP(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )(N(R P2 ) 2 ), - OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, - OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 .
  • the reactive phosphorous group is -OP(OR P )(N(R P2 ) 2 ).
  • R P is an optionally substituted C 1- 6 alkyl.
  • each R P2 is independently optionally substituted C 1-6 alkyl.
  • each R P2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl. It is noted that when two or more R P2 groups are present in the reactive phosphorous group, they can be same or different. Thus, in some none- limiting examples, when two or more R P2 groups are present, the R P2 groups are different. In some other non-limiting examples, when two or more R P2 groups are present, the R P2 groups are same. In some embodiments of any one of the aspects, each R P2 is isopropyl.
  • both R P2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl.
  • R P and one of R P2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl.
  • each R P3 is independently optionally substituted C 1-6 alkyl.
  • R P3 is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 - C 6 alkoxy.
  • the reactive phosphorous group is - OP(OR P )(N(R P2 ) 2 ).
  • the reactive phosphorous group is -OP(OR P )(N(R P2 ) 2 ), where R P is cyanoethyl (-CH 2 CH 2 CN) and each R P2 is isopropyl.
  • R 32 is - OP(OR P )(N(R P2 ) 2 ), -OP(SR P )(N(R P2 ) 2 ),-OP(O)(OR P )(N(R P2 ) 2 ), -
  • R 32 is -OP(OR P ) (N(R P2 ) 2 ) , - OP(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )(N(R P2 ) 2 ), -OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), - OP(O)(OR P )H, -OP(S)(OR P ) an optionally substituted C 1-6 alkyl, each R P2 is independently optionally substituted C 1-6 alkyl; and each R P3 is independently optionally substituted C 1-6 alkyl.
  • R 32 is -OP(OR P )(N(R P2 ) 2 ).
  • the R 32 is -OP(OR P )(N(R P2 ) 2 ), where R P is cyanoethyl (-CH 2 CH 2 CN) and each R P2 is isopropyl.
  • R 32 is a solid support or a linker covalently attached to a solid support.
  • R 32 is -OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • R 32 is -OC(O)CH 2 CH 2 CO 2 H.
  • R 322 when R 32 is -OR 322 , R 322 can be hydrogen or a hydroxyl protecting group.
  • R 323 can be hydrogen or a sulfur protecting group. Accordingly, in some embodiments of any one of the aspects, R 323 is hydrogen.
  • R 32 is - O(CH 2 CH 2 O) r CH 2 CH 2 OR 324
  • r can be 1 -50
  • R 324 is independently for each occurrence H, C 1 -C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 325
  • R 325 is independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diary lamino, heteroarylamino, or diheteroaryl amino.
  • R 32 is -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 325
  • s can be 1-50 and R 325 can be independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diary lamino, heteroarylamino, or diheteroaryl amino.
  • R 32 is hydrogen, halogen, -OR 322 , or optionally substituted C 1 -C 30 alkoxy.
  • R 32 is halogen, -OR 322 , or optionally substituted C 1 -C 30 alkoxy.
  • R 32 is F, OH or optionally substituted C 1 -C 30 alkoxy.
  • R 32 is C 1 -C 30 alkoxy optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 32 is -O(CH 2 ) t CH 3 , where t is 1-21.
  • t is 14, 15, 16, 17 or 18. In one non-limiting example, t is 16.
  • R 32 is -O(CH 2 ) u R 327 , where u is 2-10; R 327 is C 1 -C 6 alkoxy, amino (NH 2 ), CO 2 H, OH or halo.
  • R 327 is -CH 3 or NH 2 .
  • R 32 is -O(CH 2 ) u - OMe or R 32 is -O(CH 2 ) u NH 2 .
  • u is 2, 3, 4, 5 or 6.
  • u is 2, 3 or 6.
  • u is 2.
  • u is 3 or 6.
  • R 32 is a C 1 - C 6 haloalkyl.
  • R 32 is a C 1 -C 4 haloalkyl.
  • R 32 is -CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 or -CF 2 (CF 3 ) 2 .
  • R 32 is - OCH(CH 2 OR 328 )CH 2 OR 329 , where R 328 and R 329 independently are H, optionally substituted C 1 - C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 328 and R 329 independently are optionally substituted C 1 -C 30 alkyl.
  • R 32 is -CH 2 C(O)NHR 3210 , where R 3210 is H, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 3210 is H or optionally substituted C 1 -C 30 alkyl.
  • R 3210 is optionally substituted C 1 -C 6 alkyl.
  • R 33 is hydrogen, halogen, -OR 332 , -SR 333 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2 - 30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR 334 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 335 , NHC(O)R 336 , a lipid, a linker co
  • R 332 can be H, hydroxyl protecting group, optionally substituted C 1-30 alkyl, C 1- 30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 333 can be H, sulfur protecting group, optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 334 can be H, hydroxyl protecting group, optionally substituted C 1-30 alkyl, C 1- 30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 335 can be hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.
  • R 336 can be can be hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1- 3 oalkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.
  • R 33 is a reactive phosphorus group.
  • R 33 is -OP(OR P )(N(R P2 ) 2 ), -OP(SR P )(N(R P2 ) 2 ) , - OP(O)(OR P )(N(R P2 ) 2 ), -OP(S)(OR P )(N(R P2 ) 2 ) , -OP(O)(SR P )(NR P2 ) 2 , -OP(O)(OR P )H, -
  • R 33 is -OP(OR P )(N(R P2 ) 2 ), - OP(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )(N(R P2 ) 2 ), -OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), - OP(O)(OR P )H, -OP(S)(OR P ) an optionally substituted C 1-6 alkyl, each R P2 is independently optionally substituted C 1-6 alkyl; and each R P3 is independently optionally substituted C 1-6 alkyl.
  • R 33 is -OP(OR P )(N(R P2 ) 2 ).
  • the R 33 is -OP(OR P )(N(R P2 ) 2 ), where R P is cyanoethyl (-CH 2 CH 2 CN) and each R P2 is isopropyl.
  • R 32 and R 33 are a reactive phosphorous group.
  • R 33 is a solid support or a linker covalently attached to a solid support.
  • R 33 is -OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • R 32 and R 33 are a solid support or a linker covalently attached to a solid support.
  • R 332 when R 33 is -OR 332 , R 332 can be hydrogen or a hydroxyl protecting group.
  • R 332 can be hydrogen in some embodiments of any one of the aspects described herein.
  • R 33 is -OC(O)CH 2 CH 2 CO 2 H.
  • R 333 can be hydrogen or a sulfur protecting group. Accordingly, in some embodiments of any one of the aspects, R 333 is hydrogen.
  • R 33 is -O(CH 2 CH 2 O) r CH 2 CH 2 OR 334
  • r can be 1 -50
  • R 334 is independently for each occurrence H, C 1 -C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 335
  • R 335 is independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diary lamino, heteroarylamino, or diheteroaryl amino.
  • R 33 is -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 335
  • s can be 1-50 and R 335 can be independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diary lamino, heteroarylamino, or diheteroaryl amino.
  • R 33 is hydrogen, halogen, -OR 332 , or optionally substituted C 1 -C 30 alkoxy.
  • R 33 is halogen, -OR 332 , or optionally substituted C 1 -C 30 alkoxy.
  • R 33 is F, OH or optionally substituted C 1 -C 30 alkoxy.
  • R 33 is C 1 -C 30 alkoxy optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 33 is -O(CH 2 ) t CH 3 , where t is 1-21.
  • t is 14, 15, 16, 17 or 18. In one non-limiting example, t is 16.
  • R 33 is -O(CH 2 ) u R 337 , where u is 2-10; R 337 is C 1 -C 6 alkoxy, amino (NH 2 ), CO 2 H, OH or halo.
  • R 337 is -CH 3 or NH 2 .
  • R 33 is -O(CH 2 ) u - OMe or R 33 is -O(CH 2 ) u NH 2 .
  • u is 2, 3, 4, 5 or 6.
  • u is 2, 3 or 6.
  • u is 2.
  • u is 3 or 6.
  • R 33 is a C 1 - C 6 haloalkyl.
  • R 33 is a C 1 -C 4 haloalkyl.
  • R 33 is -CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 or -CF 2 (CF 3 ) 2 .
  • R 33 is - OCH(CH 2 OR 338 )CH 2 OR 339 , where R 338 and R 339 independently are H, optionally substituted C 1 - C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 338 and R 339 independently are optionally substituted C 1 -C 30 alkyl.
  • R 33 is -CH 2 C(O)NHR 3310 , where R 3310 is H, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 3310 is H or optionally substituted C 1 -C 30 alkyl.
  • R 3310 is optionally substituted C 1 -C 6 alkyl.
  • R 33 and R 4 taken together with the atoms to which they are attached form an optionally substituted C 3-8 cycloalkyl, optionally substituted C 3-8 cycloalkenyl, or optionally substituted 3-8 membered heterocyclyl.
  • R 35 is R 551 , optionally substituted C 1-6 alkyl-R 551 , optionally substituted -C 2-6 alkenyl-R 551 , or optionally substituted -C 2 - 6 alkynyl-R 551 , where R 551 can be -OR 552 , -SR 553 , hydrogen, a phosphorous group, a solid support or a linker to a solid support.
  • R 551 is -OR 552
  • R 552 can be H or a hydroxyl protecting group.
  • R 551 is -SR 553
  • R 553 can be H or a sulfur protecting group.
  • R 35 is -OR 552 or - SR 553 .
  • R 552 is a hydroxyl protecting group.
  • Exemplary hydroxyl protecting groups for R 552 include, but are not limited to, benzyl, benzoyl, 2,6-dichlorobenzyI, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityI (DMT), 9-phenylxanthine-9-yI (Pixyl) and 9-(p-methoxyphenyl)xanthine-9- yl (MOX).
  • R 35 is -OR 552 and R 552 is 4,4'-dimethoxytrityl (DMT), e.g., R 35 is -O-DMT.
  • R 35 is -CH(R 554 )- R 551 , where R 554 is hydrogen, halogen, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl, optionally substituted C 2 -C 30 alkynyl, or optionally substituted C 1 -C 30 alkoxy.
  • R 35 is -CH(R 554 )-R 551
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 35 is optionally substituted C 1-6 alkyl-R 551 or optionally substituted -C 2-6 alkenyl-R 551 ,
  • R 551 is a reactive phosphorous group.
  • each R 555 is independently hydrogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2 - 30 alkynyl, or an oxygen-protecting group; and each R 556 is independently hydrogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl, or a sulfur-protecting group.
  • At least one at least one R 555 in P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ) is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2 - 30 alkynyl, or an oxygen-protecting group.
  • At least one R 555 is H and at least one R 555 is other than H in -P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ).
  • all R 555 are H in -P(O)(OR 555 ) 2 , - P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), - OP(S)(SR 556 ) 2 , -SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 555 are other than H in in - P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , -OP(S)(OR 555 ) 2 , -
  • SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is H.
  • SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is other than H.
  • SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl, or an sulfur-protecting group.
  • At least one R 556 is H and at least one R 556 is other than H in -P(S)(SR 556 ) 2 , -OP(S)(SR 556 ) 2 and -SP(S)(SR 556 ) 2 .
  • all R 556 are H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 556 are other than H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • R 33 is a reactive phosphorous group, a solid support, a linker to a solid support, and R 35 is a protected hydroxyl.
  • R 32 is a reactive phosphorous group, a solid support, a linker to a solid support, and R 35 is a protected hydroxyl.
  • L is a linker
  • linker means an organic moiety that connects two parts of a compound.
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR 1 , C(O), C(O)O, C(O)NR', SO, SO 2 , SO 2 NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalken
  • the linker is a cleavable linker.
  • Cleavable linkers are those that rely on processes inside a target cell to liberate the two parts the linker is holding together, as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by specific enzymes (e.g. proteases) within the cell.
  • cleavable linkers allow the two parts to be released in their original form after internalization and processing inside a target cell.
  • Cleavable linkers include, but are not limited to, those whose bonds can be cleaved by enzymes (e.g., peptide linkers); reducing conditions (e.g., disulfide linkers); or acidic conditions (e.g., hydrazones and carbonates).
  • enzymes e.g., peptide linkers
  • reducing conditions e.g., disulfide linkers
  • acidic conditions e.g., hydrazones and carbonates.
  • the cleavable linker comprises at least one cleavable linking group.
  • a cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.
  • the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood or serum of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood.
  • degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
  • redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g
  • a cleavable linkage group such as a disulfide bond can be susceptible to pH.
  • the pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1- 7.3.
  • Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0.
  • Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
  • a linker can include a cleavable linking group that is cleavable by a particular enzyme.
  • the type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
  • the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • a degradative agent or condition
  • the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • the evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals.
  • useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
  • cleavable linking groups is redox cleavable linking groups, which may be used in the dsRNA molecule according to the present invention that are cleaved upon reduction or oxidation.
  • reductively cleavable linking group is a disulfide linking group (-S-S-).
  • a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein.
  • a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell.
  • DTT dithiothreitol
  • the candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions.
  • candidate compounds are cleaved by at most 10% in the blood.
  • useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).
  • the rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
  • Phosphate-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by agents that degrade or hydrolyze the phosphate group.
  • agents that degrade or hydrolyze the phosphate group are enzymes such as phosphatases in cells.
  • phosphate-based linking groups are -O-P(O)(ORk)-O-, -O- P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O- P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(
  • Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S- P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(O)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-, -O-P(S)(H)-S-.
  • a preferred embodiment is -O-P(O)(OH)-O-.
  • Acid cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are linking groups that are cleaved under acidic conditions.
  • acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid.
  • specific low pH organelles such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups.
  • acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids.
  • a preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.
  • Ester-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as esterases and amidases in cells.
  • ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups.
  • Ester cleavable linking groups have the general formula - C(O)O-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.
  • Peptide-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as peptidases and proteases in cells.
  • Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.
  • Peptide-based cleavable groups do not include the amide group (-C(O)NH-).
  • the amide group can be formed between any alkylene, alkenylene or alkynylene.
  • a peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins.
  • the peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group.
  • Peptide-based cleavable linking groups have the general formula - NHCHR A C(O)NHCHR B C(O)-, where R A and R B are the R groups of the two adjacent amino acids.
  • L is a bond
  • L is absent, e.g., R 6 or R 7 is -R L .
  • intemucleoside linkage refers to a covalent linkage between adjacent nucleosides.
  • the two main classes of intemucleoside linkages are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino ( — CH2-N(CH3)-O — CH2-), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ); siloxane ( — O — Si(H)2-O — ); and N,N'- dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-).
  • Modified intemucleoside linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide compound.
  • linkages having a chiral atom can be prepared as racemic mixtures, as separate enantiomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous- containing and non-phosphorous-containing linkages are well known to those skilled in the art.
  • the phosphate group in the intemucleoside linkage can be modified by replacing one of the oxygens with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown.
  • modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • one of the non-bridging phosphate oxygen atoms in the phosphodiester intemucleoside linkage can be replaced by any of the following: S, Se, BR 3 (R is hydrogen, alkyl, aryl), C (i.e.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral. In other words a phosphorous atom in a phosphate group modified in this way is a stereogenic center.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).
  • Phosphorodithioates have both non-bridging oxygens replaced by sulfur.
  • the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers.
  • modifications to both non-bridging oxygens, which eliminate the chiral center, e.g. phosphorodithioate formation can be desirable in that they cannot produce diastereomer mixtures.
  • the non-bridging oxygens can be independently any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).
  • a phosphodiester intemucleoside linkage can also be modified by replacement of bridging oxygen, (i.e. oxygen that links the phosphate to the sugar of the nucleosides), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • bridging oxygen i.e. oxygen that links the phosphate to the sugar of the nucleosides
  • nitrogen bridged phosphoroamidates
  • sulfur bridged phosphorothioates
  • carbon bridged methylenephosphonates
  • Modified phosphate linkages where at least one of the oxygen linked to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorous group are also referred to as “non-phosphodiester intersugar linkage” or “non-phosphodiester linker.”
  • the phosphate group can be replaced by non-phosphorus containing connectors, e.g. dephospho linkers.
  • Dephospho linkers are also referred to as non- phosphodiester linkers herein. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.
  • Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amides, carbamate and ethylene oxide linker.
  • a modification of a non-bridging oxygen can necessitate modification of 2’-OH, e.g., a modification that does not participate in cleavage of the neighboring intersugar linkage, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA.
  • Preferred non-phosphodiester intemucleoside linkages include phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Sp isomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Rp isomer, phosphorodithioates, phsophotriesters, aminoalkylphosphotrioesters, alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidate), and boranophosphonates .
  • the oligonucleotides described herein comprise one or more neutral intemucleoside linkages that are non-ionic.
  • the non-phosphodiester backbone linkage is selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate and phosphoramidate backbone linkages.
  • the intemucleoside linkage is where R IL1 and R IL2 are each independently for each occurrence absent, O, S, CH 2 , NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, wherein backbone of the alkylene can comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and/or at the end; and R IL3 and R IL4 are each independently selected from the group consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR 3 (R is hydrogen, alkyl, aryl), BH 3 - , C (i.e.
  • R IL1 and R IL2 are replacing the oxygen linked to 5’ carbon of a first nucleoside sugar and the other of R IL1 and R IL2 is replacing the oxygen linked to 3’ (or 2’) carbon of a second nucleoside sugar.
  • R IL1 , R IL2 , R IL3 and R IL4 all are O.
  • R IL1 and R IL2 are O and at least one of R IL3 and R IL4 is other than O.
  • one of R IL3 and R IL4 is S and the other is O or both of R IL3 and R IL4 are S.
  • one of R 3 or R 5 is a bond to a modified intemucleoside linkage, e.g., an intemucleoside linkage of structure: where at least one of R IL1 , R IL2 , R IL3 and R IL4 is not O.
  • R IL3 and R IL4 is S.
  • both of R 3 and R 5 are a bond to a modified intemucleoside linkage.
  • R 3 is a bond to phosphodiester intemucleoside linkage.
  • R 5 is a bond to phosphodiester intemucleoside linkage.
  • R 3 is a bond to a modified intemucleoside linkage and R 5 is a bond to phosphodiester intemucleoside linkage.
  • R 5 is a bond to a modified intemucleoside linkage and R 3 is a bond to phosphodiester intemucleoside linkage.
  • the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more modified intemucleoside linkages.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5 or 6 modified intemucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3 or 4 modified intemucleoside linkages.
  • the oligonucleotide comprises at least two modified intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the oligonucleotide and further comprises at least two modified intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the oligonucleotide.
  • the oligonucleotide comprises modified intemucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the oligonucleotide.
  • the modified intemucleoside linkage is a phosphorothioate.
  • the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate intemucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3, 4, 5 or 6 phosphorothioate intemucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3 or 4 phosphorothioate intemucleoside linkages.
  • the oligonucleotide comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the oligonucleotide and further comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the oligonucleotide.
  • the oligonucleotide comprises modified intemucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the oligonucleotide.
  • Oxygen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • oxygen protecting groups include, but are not limited to, methyl, t- butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM), methylthiomethyl (MTM), t- butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexy
  • oxygen protecting group is benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX).
  • DMT 4,4'-dimethoxytrityl
  • Pixyl 9-phenylxanthine-9-yl
  • MOX 9-(p- methoxyphenyl)xanthine-9-yl
  • the hydroxyl protecting group is selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4 '-dimethoxytrityl.
  • protected hydroxyl and “protected hydroxy” as used herein mean a group of the formula -OR Pro , wherein R Pro is an oxygen protecting group as defined herein.
  • Nitrogen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • 2.4-dimethylthiophenyl carbamate Bmpc
  • 2- phosphonioethyl carbamate Peoc
  • 2- triphenylphosphonioisopropyl carbamate Ppoc
  • 1,1- dimethyl-2-cyanoethyl carbamate m- chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)- 6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate,
  • Additional exemplary nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N - phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N- 2,3- diphenylmaleimide, N-2,5-dimethylpyrrole, N-l,l,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3- dibenzyl-l,3,5-triazacyclohexan-2-
  • Sulfur protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • nucleoside of Formula (I) or (II) can be located anywhere in the oligonucleotide. In some embodiments, the nucleoside of Formula (I) or (II) is present at the 5’- or 3 ’-terminus of the oligonucleotide. In some embodiments, the nucleoside of Formula (I) or (II) is present at an internal position of the oliogunculeotide.
  • the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (I) or (II), a nucleoside with a modified sugar.
  • a “modified sugar” is meant a sugar or moiety other than 2’-deoxy (i.e, 2’-H) or 2 ’-OH ribose sugar.
  • nucleotides comprising a modified sugar are 2’-F ribose, 2’-OMe ribose, 2’-O,4’-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5- anhydrohexitol nucleic acid, HNA), cyclohexene (Cyclohexene nucleic acid, CeNA), 2’- methoxy ethyl ribose, 2’-O-allyl ribose, 2’-C-allyl ribose, 2'-O-N-methylacetamido (2-O-NMA) ribose, a 2'-O-dimethylaminoethoxyethyl (2-O-DMAEOE) ribose, 2'-O-aminopropyl (2-O-AP) ribose, 2’-F arabinose (2'-ara-F),
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro (2’-F) nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 102’-F nucleotides. It is noted that the 2’-F nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises 2’- nucleosides of Formula (I)/(II) and 2’-F nucleosides.
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2’-OMe nucleotides. It is noted that the 2’-OMe nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises solely comprises solely comprises 2’- nucleosides of Formula (I)/(II) and 2’-OMe nucleosides. In some other embodiments, the oligonucleotide comprises, e.g., solely comprises solely comprises 2’- nucleosides of Formula (I)/(II), 2’-OMe nucleosides and 2’-F nucleosides.
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-deoxy, e.g., 2’-H nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of 2’-deoxy, e.g., 2’-H nucleotides. It is noted that the 2’- deoxy, e.g., 2’-H nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide can comprise a 2’-deoxy, e.g., 2’-H nucleotide at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the oligonucleotide.
  • the oligonucleotide comprises a 2’-deoxy nucleotide at positions 5 and 7, counting from 5 ’-end of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises solely comprises nucleosides of Formula (I)/(II) and 2’-deoxy (2’-H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (I)/(II), 2’-OMe nucleosides, and 2’-deoxy (2’-H) nucleotides.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (I)/(II), 2’-F nucleosides and 2’-deoxy (2’-H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (I)/(II), 2’-OMe nucleosides, 2’-F nucleosides and 2’-deoxy (2’-H) nucleotides.
  • the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (I) or (II), a non-natural nucleobase.
  • the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising an independently selected non-natural nucleobase.
  • a nucleotide comprising a non-natural nucleobase can be present anywhere in the oligonucleotide.
  • non-natural nucleobase a nucleobase other than adenine, guanine, cytosine, uracil, or thymine.
  • exemplary non-natural nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(pseudouracil),
  • purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, content of all which is incorporated herein by reference.
  • the non-natural nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8- (hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopen
  • a non-natural nucleobase is a modified nucleobase, i.e., the nucleobase comprises a nucleobase modification described herein, e.g., the nucleobase is a substituted or modified analog of any of the natural nucleobases.
  • nucleobase modifications include, but not limited to: C-5 pyrimidine with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities, N 2 - and N 6 - with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities of purines, G-clamps, guanidinium G-clamps, and pseudouridine known in the art.
  • the non-natural nucleobase is a universal nucleobase.
  • a universal nucleobase is any modified or unmodified natural or non-natural nucleobase that can base pair with all of adenine, cytosine, guanine and uracil without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide comprising the universal nucleobase.
  • Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza- 7-deazaadenine, 4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6- methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4- methylinolyl, 4,6-dimethylindolyl, phenyl, nap
  • the non-matural nucleobase is a protected nucleobase.
  • a “protected nucleobase” referes to a nucleobase comprising a nitrogen protecting group, and/or an oxygen protecting group, and/or a sulfur protecting group.
  • the non-natural nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.
  • the oligonucleotide further comprises a solid support linked thereto.
  • the oligonucleotides described herein can range from few nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) in length to hunderes of nucleotides in length.
  • the oligonucleotide can be from 5 nucleotides to 100 nucleotides in length.
  • the oligonucleotide is from 10 nucleotides to 50 nucleotides in length.
  • the oligonucleotide is between 15 and 35, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length.
  • oligonucleotide In some embodiments, longer oligonucleotides of between 25 and 30 nucleotides in length are preferred. In some embodiments, shorter oligonucleotides of between 10 and 15 nucleotides in length are preferred. In another embodiment, the oligonucleotide is at least 21 nucleotides in length.
  • the oligonucleotide described herein comprises a pattern of backbone chiral centers.
  • a common pattern of backbone chiral centers comprises at least 5 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 6 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 7 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 8 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 9 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 19 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration, and no more than 8 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration, and no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration, and no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration, and no more than 4 intemucleotidic linkages which are not chiral.
  • the intemucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages which are not chiral are optionally contiguous or not contiguous.
  • the oligonucleotide described herein comprises a stereochemistry block.
  • a block is an Rp block in that each intemucleotidic linkage of the block is Rp.
  • a 5 ’-block is an Rp block.
  • a 3 ’-block is an Rp block.
  • a block is an Sp block in that each intemucleotidic linkage of the block is Sp.
  • a 5 ’-block is an Sp block.
  • a 3 ’-block is an Sp block.
  • provided oligonucleotides comprise both Rp and Sp blocks.
  • provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each intemucleotidic linkage in a natural phosphate linkage.
  • the oligonculeotide described herein comprises a 5 ’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification.
  • a 5 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’- block is an Sp block wherein each of intemucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5 ’-block comprises 4 or more nucleoside units.
  • a 5 ’-block comprises 5 or more nucleoside units. In some embodiments, a 5 ’-block comprises 6 or more nucleoside units. In some embodiments, a 5 ’-block comprises 7 or more nucleoside units. In some embodiments, a 3 ’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3’-block comprises 4 or more nucleoside units.
  • a 3 ’-block comprises 5 or more nucleoside units.
  • a 3 ’-block comprises 6 or more nucleoside units.
  • a 3 ’-block comprises 7 or more nucleoside units.
  • oligonucleotide described herein comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of intemucleotidic linkage, e.g., natural phosphate linkage, modified intemucleotidic linkage, Rp chiral intemucleotidic linkage, Sp chiral intemucleotidic linkage, etc.
  • A is followed by Sp.
  • A is followed by Rp.
  • A is followed by natural phosphate linkage (PO).
  • U is followed by Sp.
  • U is followed by Rp.
  • U is followed by natural phosphate linkage (PO).
  • C is followed by Sp.
  • C is followed by Rp.
  • C is followed by natural phosphate linkage (PO).
  • G is followed by Sp.
  • G is followed by Rp.
  • G is followed by natural phosphate linkage (PO).
  • C and U are followed by Sp.
  • C and U are followed by Rp.
  • C and U are followed by natural phosphate linkage (PO).
  • a and G are followed by Sp.
  • a and G are followed by Rp.
  • the oligonucleotides described herein are 5’ phosphorylated or include a phosphoryl analog at the 5’ prime terminus.
  • 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5'-monophosphate ((HO) 2 (O)P-O-5'); 5'-diphosphate ((HO) 2 (O)P- O-P(HO)(O)-O-5'); 5'-triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'- adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'- (HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)P- O-5'); 5
  • 5'-alpha-thiotriphosphate 5'-gamma-thiotriphosphate, etc.
  • 5'- phosphoramidates ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)P-O-5'), 5'-alkylphosphonates (e.g.,
  • exemplary 5 ’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO) 2 (X)P-O[-(CH 2 ) a -O-P(X)(OH)-O] b - 5', ((HO)2(X)P-O[-(CH 2 )
  • the oligonucleotide comprises a 5’-vinylphosphonate group.
  • the oligonucleotide comprises a 5’-E-vinyl phosphonate group.
  • the oligonucleotide comprises a 5’-Z- vinylphosphonate group.
  • the oligonucleotide dscribed herein comprises a 5’-morpholino, a 5’-dimethylamino, a 5’-deoxy, an inverted abasic, or an inverted abasic locked nucleic acid modification at the 5 ’-end.
  • the oligonucleotide dscribed herein can comprise a thermally destabilizing modification.
  • the oligonucleotide can comprise at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5 ’-end of the oligonucleotide.
  • the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5 ’-end of the antisense strand.
  • thermally destabilizing modification is located in positions 2-9, or preferably positions 4-8, counting from the 5 ’-end of the oligonucleotide.
  • the thermally destabilizing modification is located at position 5, 6, 7 or 8, counting from the 5 ’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 7, counting from the 5 ’-end of the oligonucleotide.
  • the term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5 ’-end of the antisense strand.
  • the thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).
  • UUA unlocked nucleic acids
  • GNA glycol nucleic acid
  • the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA.
  • the destabilizing modification mUNA is selected from the group consisting of
  • R H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-nPr; O- alkyl; O-alkylamino;
  • R' H, Me
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; 06-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; and
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers.
  • the destabilizing modification mUNA is selected from the group consisting of
  • R H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-nPr; O- alkyl; O-alkylamino;
  • R' H, Me
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; and
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers.
  • the destabilizing modification mUNA is selected from the group consisting of
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; 06-alkyl-G; 7-deazapurines; and Stereochemistry is R or S and combination of R and S for the unspecified chiral centers.
  • the destabilizing modification mUNA is selected from the group consisting of
  • R H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-nPr; O- alkyl; O-alkylamino;
  • R’ H, Me
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; 06-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; and
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers
  • the destabilizing modification mUNA is selected from the group consisting of
  • R H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-nPr; O- alkyl; O-alkylamino;
  • R’ H, Me
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-cIamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-aIkyI-A; 06-alkyI-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-cIamp; non-canonical mono, bi and tricyclic heterocycles; and
  • Stereochemistry is R or S and combination of R and S for the unspecified chiral centers
  • the modification mUNA is selected from the group consisting of
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-cIamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyI-A; 06-alkyI-G; 7-deazapurines; and Stereochemistry is R or S and combination of R and S for the unspecified chiral centers
  • Exemplary abasic modifications include, but are not limited to the following:
  • R H, Me, Et or OMe
  • R’ H, Me, Et or OMe
  • R” H, Me, Et or OMe
  • B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.
  • Exemplified sugar modifications include, but are not limited to the following: wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.
  • the thermally destabilizing modification of the duplex is selected from the mUNA and GNA building blocks described in Examples 1-3 herein.
  • the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA.
  • the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5 ’-Me, Hy p-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h’GNA (Mod A-Mod K).
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., Cl’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or Cl’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., Cl’, C2’, C3’, C4’ or 04’) are independently or in combination absent from the nucleotide.
  • bonds between the ribose carbons e.g., Cl’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or Cl’-O4’
  • ribose carbons or oxygen e.g., Cl’, C2’, C3’, C4’ or 04’
  • acyclic nucleotide is or wherein B is a modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • R1 and R2 independently are H, halogen, OR3, or alkyl
  • R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar.
  • the term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue.
  • UNA also encompasses monomers with bonds between C1'-C4' being removed (i.e. the covalent carbon- oxygen-carbon bond between the Cl' and C4' carbons).
  • the C2-C3' bond i.e. the covalent carbon-carbon bond between the C2' and C3' carbons
  • the acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings.
  • the acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.
  • glycol nucleic acid refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:
  • the thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex.
  • Exemplary mismatch base pairs Other mismatch base pairings known in the art are also amenable to the present invention.
  • a mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides.
  • the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand.
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as:
  • the thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.
  • the thermally destabilizing modification includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand.
  • nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety.
  • Exemplary nucleobase modifications are:
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more a-nucleotide complementary to the base on the target mRNA, such as: wherein R is H, OH, OC3 ⁇ 4, F, NH 2 , NHMe, NMe 2 or O-alkyl
  • the alkyl for the R group can be a C 1 -C 6 alkyl.
  • Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.
  • the oligonucleotide can comprise one or more stabilizing modifications.
  • the oligonucleotide can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • the oligonucleotide comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • a stabilizing modification in the oligonucleotide can be present at any positions.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16, counting from the 5 ’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 6, 14 and 16, counting from the 5 ’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 14 and 16, counting from the 5 ’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 7, 10 and 11, counting from the 5’-end. In some other embodiments, the oligonucleotide comprises stabilizing modifications at positions 7, 9, 10 and 11, counting from the 5 ’-end.
  • the oligonucleotide comprises at least one stabilizing modification adjacent to a destabilizing modification.
  • the stabilizing modification can be the nucleotide at the 5 ’-end or the 3 ’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification.
  • the oligonucleotide comprises a stabilizing modification at each of the 5 ’-end and the 3 ’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the oligonucleotide comprises at least two stabilizing modifications at the 3 ’-end of a destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • thermally stabilizing modifications include, but are not limited to 2’-fluoro modifications.
  • Other thermally stabilizing modifications include, but are not limited to LNA.
  • oligonucleotides described herein can be used for any use knonw in the art for oligonucleotides.
  • the oligonucleotides described herein can be used RNA interference based gene silencing techniques.
  • Some exemplary uses for the oligonucleotides described herein include, but are not limited to, RNA interference agents, antisense oligonucelotides, aptamers, miRNAs, ribozymes, triplex forming oligonucleotides and the like.
  • an oligonucleotide described herein is comprised in a double-stranded nucleic acid.
  • an olignucloetide described herein can be one strand of a dsRNA molecule.
  • the dsRNA molecule comprises a sense strand (also referred to as passenger strand) and an antisense strand (also referred to as guide strand).
  • An oligonucleotide described herein can be used as the sense and/or the antisense strand of the dsRNA molecule.
  • an oligonucleotide described herein is the sense strand of the dsRNA molecule.
  • an oligonucleotide described herein is the antisense strand of the dsRNA molecule.
  • Each strand of the dsRNA molecule can range from 15-35 nucleotides in length.
  • each strand can be between, 17-35 nucleotides in length, 17-30 nucleotides in length, 25- 35 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19- 21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.
  • the sense and antisense strands can be equal length or unequal length.
  • the sense strand and the antisense strand independently have a length of 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
  • the antisense strand is of length 15-35 nucleotides. In some embodiments, the antisense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19- 25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length.
  • the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.
  • the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length.
  • the antisense strand is 22, 23 or 24 nucleotides in length.
  • the antisense strand is 23 nucleotides in length.
  • the sense strand can be, in some embodiments, 15-35 nucleotides in length. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 25-35, 27- 30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides in length. In some particular embodiments, the sense strand is 20, 21 or 22 nucleotides in length. In example, the sense strand is 21 nucleotides in length
  • the sense strand can be 15-35 nucleotides in length, and the antisense strand can be independent from the sense strand, 15-35 nucleotides in length.
  • the sense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length
  • the antisense strand is independently 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21- 25, 21-25, or 21-23 nucleotides in length.
  • the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.
  • the sense strand and the antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 19, 20, 21, 22 or 23 nucleotides in length and the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 20, 21 or 22 nucleotides in length and the antisense strand is 22, 23 or 24 nucleotides in length.
  • the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.
  • the sense strand and antisense strand typically form a double-stranded or duplex region.
  • the duplex region of a dsRNA agent described herein can be 12-35 nucleotide (or base) pairs in length.
  • the duplex region can be between 14-35 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotides in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19- 21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length.
  • the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.
  • the duplex region is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length.
  • the duplex region is 19, 20, 21, 22 or 23 nucleotide pairs in length.
  • the duplex region is 20, 21 or 22 nucleotide pairs in length.
  • the dsRNA molecule has a duplex region of 21 base pairs.
  • the oligonucleotide described herein or the antisense strand of the dsRNA molecule described herein comprises a nucleotide sequence substantially complementary to a target nucleic acid, e.g., a target gene or mRNA.
  • the disclosure is directed to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene.
  • the present invention further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in vitro.
  • the disclosure is directed to a use of an oligonucleotide and/or dsRNA molecule described herein for use in inhibiting expression of a target gene in a subject.
  • the subject may be any animal, such as a mammal, e.g., a mouse, a rat, a sheep, a cattle, a dog, a cat, or a human
  • the oligonucleotide and/or dsRNA molecule described herein is administered in buffer.
  • oligonucleotide and/or dsRNA molecule described herein described herein can be formulated for administration to a subject.
  • a formulated oligonucleotide and/or dsRNA composition can assume a variety of states.
  • the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water).
  • the siRNA is in an aqueous phase, e.g., in a solution that includes water.
  • the aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • a delivery vehicle e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • the siRNA composition is formulated in a manner that is compatible with the intended method of administration, as described herein.
  • the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
  • a oligonucleotide and/or dsRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide and/or dsRNA, e.g., a protein that complexes with oligonucleotide and/or dsRNA.
  • another agent e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide and/or dsRNA, e.g., a protein that complexes with oligonucleotide and/or dsRNA.
  • Still other agents include chelating agents, e.g., EDTA (e.g., to remove divalent cations such as Mg 2+ ), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
  • the oligonucleotide and/or dsRNA preparation includes another dsRNA compound, e.g., a second dsRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene.
  • another dsRNA compound e.g., a second dsRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene.
  • Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different siRNA species.
  • Such dsRNAs can mediate RNAi with respect to a similar number of different genes.
  • the oligonucleotide and/or dsRNA preparation includes at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA).
  • a second therapeutic agent e.g., an agent other than a RNA or a DNA.
  • a oligonucleotide and/or dsRNA composition for the treatment of a viral disease e.g., HIV
  • a known antiviral agent e.g., a protease inhibitor or reverse transcriptase inhibitor
  • a dsRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.
  • Liposomes A oligonucleotide and/or dsRNA preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle.
  • liposome refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the oligonucleotide and/or dsRNA composition.
  • the lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the oligonucleotide and/or dsRNA composition, although in some examples, it may.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the oligonucleotide and/or dsRNA are delivered into the cell where the dsRNA can specifically bind to a target RNA and can mediate RNAi. In some embodiments, the liposomes are also specifically targeted, e.g., to direct the oligonucleotide and/or dsRNA to particular cell types.
  • a liposome containing oligonucleotide and/or dsRNA can be prepared by a variety of methods.
  • the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component.
  • the lipid component can be an amphipathic cationic lipid or lipid conjugate.
  • the detergent can have a high critical micelle concentration and may be nonionic.
  • Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine.
  • the dsRNA preparation is then added to the micelles that include the lipid component.
  • the cationic groups on the lipid interact with the siRNA and condense around the dsRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of oligonucleotide and/or dsRNA.
  • a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition.
  • the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favor condensation.
  • Liposome formation can also include one or more aspects of exemplary methods described in Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413- 7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, etal. Biochim. Biophys.
  • Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, which is incorporated by reference in its entirety). These methods are readily adapted to packaging oligonucleotide and/or dsRNA preparations into liposomes.
  • Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated by reference in its entirety).
  • liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Examples of other methods to introduce liposomes into cells in vitro include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.
  • cationic liposomes are used.
  • Cationic liposomes possess the advantage of being able to fuse to the cell membrane.
  • Non-cationic liposomes although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver siRNAs to macrophages.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated siRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245).
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • a positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of siRNA (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No.
  • a DOTMA analogue, 1 ,2-bis(oleoyloxy)-3-(trimethylammonia)propane can be used in combination with a phospholipid to form DNA-complexing vesicles.
  • LipofectinTM Bethesda Research Laboratories, Gaithersburg, Md. is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes.
  • DOTAP cationic lipid, l,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane
  • cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (TransfectamTM, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
  • DOGS 5-carboxyspermylglycine dioctaoleoylamide
  • DPES dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide
  • Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991, which is incorporated by reference in its entirety).
  • these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions.
  • Other commercially available cationic lipid products include DMRIE and DMRIE- HP (Vical, La Jolla, California) andLipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland).
  • DOSPA Lipofectamine
  • Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
  • Liposomes are particularly suited for topical administration. Liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer siRNA, into the skin.
  • liposomes are used for delivering siRNA to epidermal cells and also to enhance the penetration of siRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/ cholesterol/polyoxyethylene- 10-stearyl ether) were used to deliver a drug into the dermis of mouse skin.
  • Such formulations with dsRNA descreibed herein are useful for treating a dermatological disorder.
  • Liposomes that include oligonucleotide and/or dsRNA described herein can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome.
  • transfersomes are a type of deformable liposomes. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include oligonucleotide and/or dsRNA described herein can be delivered, for example, subcutaneously by infection in order to deliver dsRNA to keratinocytes in the skin.
  • lipid vesicles In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transfersomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self- repairing, and can frequently reach their targets without fragmenting, and often self-loading. [00272] Other formulations amenable to the present invention are described in United States provisional application serial nos.
  • the oligonucleotide and/or dsRNA compositions can include a surfactant.
  • the dsRNA is formulated as an emulsion that includes a surfactant.
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • Micelles and other Membranous Formulations are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
  • a mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the oligonucleotide and/or dsRNA composition, an alkali metal C 8 to C 22 alkyl sulphate, and a micelle forming compounds.
  • Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.
  • the micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide
  • a first micellar composition which contains the oligonucleotide and/or dsRNA composition and at least the alkali metal alkyl sulphate.
  • the first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition.
  • the micellar composition is prepared by mixing the dsRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
  • Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth.
  • phenol and/or m-cresol may be added with the micelle forming ingredients.
  • An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
  • micellar formulation For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant.
  • the propellant which is under pressure, is in liquid form in the dispenser.
  • the ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve.
  • the dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
  • Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether.
  • HFA 134a (1,1, 1,2 tetrafluoroethane) may be used.
  • dsRNA preparations can be incorporated into a particle, e.g., a microparticle.
  • Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.
  • the oligonucleotide and/or dsRNA described herein can be formulated for pharmaceutical use.
  • the present invention further relates to a pharmaceutical composition comprising the oligonucleotide and/or dsRNA described herein.
  • Pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the dsRNA molecules in any of the preceding embodiments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.
  • terapéuticaally-effective amount means that amount of a compound, material, or composition comprising a dsRNA molecule described herein which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention.
  • an aforementioned formulation renders orally bioavailable a compound of the present invention.
  • Methods of preparing these formulations or compositions include the step of bringing into association an oligonucleotide and/or dsRNA with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • the rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally- administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • treatment is intended to encompass therapy and cure.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • the oligonucleotide and/or dsRNA described herein or a pharmaceutical composition comprising an oligonucleotide and/or dsRNA described herein can be administered to a subject using different routes of delivery.
  • a composition that includes an oligonucleotide and/or dsRNA described herein described herein can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
  • routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
  • the oligonucleotide and/or dsRNA described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
  • the route and site of administration may be chosen to enhance targeting.
  • Lung cells might be targeted by administering the oligonucleotide and/or dsRNA described herein in aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the oligonucleotide and/or dsRNA described herein and mechanically introducing the oligonucleotide and/or dsRNA described herein.
  • a method of administering an oligonucleotide and/or dsRNA described herein, to a subject e.g., a human subject.
  • the present invention relates to an oligonucleotide and/or dsRNA described herein for use in inhibiting expression of a target gene in a subject.
  • the method or the medical use includes administering a unit dose of the oligonucleotide and/or dsRNA described herein.
  • the unit dose is less than 10 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g., about 4.4 x 10 16 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of oligonucleotide and/or dsRNA described herein per kg of bodyweight.
  • RNA agent e.g., about 4.4 x 10 16 copies
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target gene.
  • the unit dose for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application.
  • dosages may be less than 10, 5, 2, 1, or 0.1 mg/kg of body weight.
  • the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days.
  • the unit dose is not administered with a frequency (e.g., not a regular frequency).
  • the unit dose may be administered a single time.
  • the effective dose is administered with other traditional therapeutic modalities.
  • a subject is administered an initial dose and one or more maintenance doses.
  • the maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half less of the initial dose.
  • a maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 pg to 15 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day.
  • the maintenance doses are, for example, administered no more than once every 2, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient.
  • the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days.
  • the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracistemal or intracapsular), or reservoir may be advisable.
  • a delivery device e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracistemal or intracapsular), or reservoir may be advisable.
  • the composition includes a plurality of dsRNA molecule species.
  • the dsRNA molecule species has sequences that are nonoverlapping and non-adjacent to another species with respect to a naturally occurring target sequence.
  • the plurality of dsRNA molecule species is specific for different naturally occurring target genes.
  • the dsRNA molecule is allele specific.
  • the administration of the oligonucleotide and/or dsRNA composition described herein is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • the invention provides methods, compositions, and kits, for rectal administration or delivery of oligonucleotide and/or dsRNA composition described herein.
  • aspects of the disclosure also relate to methods for inhibiting the expression of a target gene.
  • the method comprises administering to the subject in an amount sufficient to inhibit expression of the target gene: (i) a double-stranded RNA described herein, where the wherein the first strand is complementary to a target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to a target gene.
  • the present disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell.
  • the present disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell in vitro.
  • the invention relates to a method of modulating the expression of a target gene in a cell, comprising administering to said cell an oligonucleotide and/or dsRNA molecule described herein.
  • the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu
  • Embodiment 1 A method for preparing an oligonucleotide comprising a nucleoside of Formula (I), the method comprising reacting an oligonucleotide comprising a nucleoside of Formula (II) with an amine of formula HNR 6 R 7 , wherein: R H is halogen (e.g., chloro or fluoro); R 2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C 4-30 alkyl-ON(CH 2 R 8
  • Embodiment 2 The method of claim 1, wherein R H is fluoro.
  • Embodiment 3 The method of claim 1 or 2, wherein at least one L is a bond.
  • Embodiment 4 The method of any one of claims 1-3, wherein at least one L is a linker.
  • Embodiment 5 The method of any one of claims 1-4, wherein at least one R L is selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucelsides and nucleotides, oligonucleotides, detectable labels, diagnostic agents (e.g., bitoin), fluorescent dyes, polyethylene glycols (PEGs), antibodies, antibody fragments (e.g., nanobodies).
  • diagnostic agents e.g., bitoin
  • fluorescent dyes e.g., polyethylene glycols (PEGs)
  • PEGs polyethylene glycols
  • antibodies antibody fragments (e.g., nanobodies).
  • Embodiment 6 The method of any one of claims 1-5, wherein at least one R L is a ligand selected from targeting ligands, endosomolytic ligands and PK modulating ligands.
  • Embodiment 7 The method of any one of claims 1-6, wherein R 6 and R 7 are same.
  • Embodiment 8 The method of any one of claims 1-7, wherein R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-6 alkoxy (e.g., methyl) or alkoxyalkyl (e.g. 2-methoxyethyl); or R 4 and R 2 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y- C(R 10 R 11 ) v -2’.
  • Embodiment 9 The method of any one of claims 1-8, wherein R 2 is hydrogen, hydroxyl, F, methoxy, or R 4 and R 2 taken together are 4’-C(R 10 R 11 ) v -Y-2’.
  • Embodiment 10 The method of any one claims 1-9, wherein R 4 and R 2 taken together are 4’-C(R 10 R 11 )vY-2’.
  • Embodiment 11 The method of any one of claims 1-10, wherein R 4 is H.
  • Embodiment 12 The method of any one of claims 1-11, wherein R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, hydroxy, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), amino, alkylamino, dialkylamino, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a ligand, a linker covalently bonded to one or more ligands (e.g., N-acetylgalactosamine (GalNac)), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • alkoxyalkyl e.g., methoxyethyl
  • amino al
  • Embodiment 13 The method of any one of claims 1-12, wherein R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, hydroxy, optionally substituted C 1-30 alkoxy, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • a 3’-oligonuclotide capping group e.g., an inverted nucleotide or an inverted abasic nucleotide
  • a linker covalently bonded e.g., -C(O)CH 2 CH 2 C(O)-
  • Embodiment 14 The method of any one of claims 1-13, wherein R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide.
  • Embodiment 15 The method of any one of claims 1-14, wherein R 3 is hydroxyl.
  • Embodiment 16 The method of any one of claims 1-15, wherein R 5 is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxy, optionally substituted C 1-30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates and phosphate mimics.
  • VP vinylphosphonate
  • Embodiment 17 The method of any one of claims 1-16, wherein R 5 is a bond to an intemucleotide linkage to a preceding nucleotide.
  • Embodiment 18 The method of any one of claims 1-17, wherein R 5 is hydroxy, optionally substituted C 1-30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate.
  • VP vinylphosphonate
  • Embodiment 19 The method of any one of claims 1-18, wherein the oligonucleotide comprises from 3 to 50 nucleotides.
  • Embodiment 20 The method of any one of claims 1-19, wherein the oligonucleotide comprises at least one ribonucleotide.
  • Embodiment 21 The method of any one of claims 1-20, wherein the oligonucleotide comprises at least one 2’-deoxyribonucleotide.
  • Embodiment 22 The method of any one of claims 1-21, wherein the oligonucleotide comprises at least one nucleotide with a modified or non-natural nucleobase.
  • Embodiment 23 The method of any one of claims 1-22, wherein the oligonucleotide comprises at least one nucleotide with a modified ribose sugar.
  • Embodiment 24 The method of any one of claims 1-23, wherein the oligonucleotide comprises at least one nucleotide comprising a group other than H or OH at the 2 ’-position of the ribose sugar.
  • Embodiment 25 The method of any one of claims 1-24, wherein the oligonucleotide comprises at least one nucleotide with a 2’-F ribose.
  • Embodiment 26 The method of any one of claims 1-25, wherein the oligonucleotide comprises at least one nucleotide with a 2’-OMe ribose.
  • Embodiment 27 The method of any one of claims 1-26, wherein the oligonucleotide comprises at least one nucleotide comprising a moiety other than a ribose sugar.
  • Embodiment 28 The method of any one of claims 1-27, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.
  • Embodiment 29 The method of any one of claims 1-28, wherein the oligonucleotide comprises at least 2, e.g., 3, 4 or 5 consecutive independently selected monomers of the Formula (I) and/or (II).
  • Embodiment 30 The method of any one of claims 1-29, wherein the oligonucleotide is attached to a solid support.
  • Embodiment 31 The method of any one of claims 1-30, wherein the oligonucleotide comprises at least one hydroxyl, phosphate or amino protecting group.
  • Embodiment 32 An oligonucleotide prepared by a method of any one of claims 1-31.
  • Embodiment 33 An oligonucleotide comprising a nucleoside of Formula (II).
  • Embodiment 34 A compound of Formula (III), wherein: R H is halogen; R 32 is hydrogen, hydroxy, halogen protected hydroxy, phosphate group, reactive phosphorous group , optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2 - 30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy), , alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl- ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-)
  • Embodiment 35 The compound of claim 34, wherein R H is fluoro.
  • Embodiment 36 The compound of claim 34 or 35, wherein R 32 is halogen; or R 4 and
  • R 32 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y-C(R 10 R 11 ) v -2’.
  • Embodiment 37 The compound of any one of claims 34-36, wherein R 32 is F, or R 4 and R 2 taken together are 4’-C(R 10 R 11 ) v -Y-2’.
  • Embodiment 38 The compound of any one claims 34-37, wherein R 4 and R 32 taken together are 4’-C(R 10 R 11 ) v -Y-2’.
  • Embodiment 39 The compound of any one of claims 34-37, wherein R 4 is H.
  • Embodiment 40 The compound of any one of claims 34-39, wherein R 33 is hydroxy, protected hydroxy, a phosphate group, a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • Embodiment 41 The compound of any one of claims 34-40, wherein R 33 is hydroxy, a phosphate group, a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 33 is hydroxy, a phosphate group, a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • Embodiment 42 The compound of any one of claims 34-41, wherein R 35 is hydroxy, protected hydorxy, optionally substituted C 1-30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma- thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates and phosphate mimics.
  • VP vinylphosphonate
  • Embodiment 43 The compound of any one of claims 34-42, wherein R 35 is hydroxyl or protected hydroxyl.
  • Embodiment 44 The compound of any one of claims 34-43, wherein R 33 is a reactive phosphorous group and R 35 is a protected hydroxyl.
  • Embodiment 45 The compound of any one of claims 34-43, wherein R 32 is a reactive phosphorous group and R 35 is a protected hydroxyl.
  • Embodiment 46 The compound of any one of claims 34-41, wherein R 35 is a vinylphosphonate (VP) group, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic; and R 33 is a reactive phosphorous group.
  • VP vinylphosphonate
  • R 33 is a reactive phosphorous group.
  • Embodiment 47 The compound of any one of claims 34-41, wherein R 35 is a vinylphosphonate (VP) group, cyclopropylphosphonate, or a phosphate mimic; andR 33 is a reactive phosphorous group.
  • R 35 is a vinylphosphonate (VP) group, cyclopropylphosphonate, or a phosphate mimic
  • R 33 is a reactive phosphorous group.
  • Embodiment 48 The compound of any one of claims 34-41, wherein R 35 is a vinylphosphonate (VP) group (e.g., E- vinylphosphonate), cyclopropylphosphonate, or a phosphate mimic; and R 33 is a phosphoramidite group.
  • VP vinylphosphonate
  • Embodiment 49 The compound of any one of claims 34-41, wherein R 35 is a triphosphate group and R33 is allyloxy, azidomethoxy, or aminooxy.
  • Embodiment 50 The compound of claim 34, wherein the compound is
  • Embodiment 51 The compound of claim 50, wherein R 33 is a reactive phosphorous group.
  • Embodiment 52 A method for preparing an oligonucleotide comprising a nucleoside of Formula (X), the method comprising reacting an oligonucleotide comprising a nucleoside of Formula (XI) with an alkali hydroxide or alkali earth hydroxide (e.g., NaOH), wherein: R H is halogen (e.g., chloro or fluoro); R 2 is hydrogen, hydroxy, protected hydroxy, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C 4-30
  • R H is
  • Embodiment 54 method of claim 52 or 53, wherein the oligonucleotide comprising a nucleoside of formula (XI) is linked to a solid support and the oligonucleotide comprising a nucleoside of formula (X) is not linked to a solid support.
  • Embodiment 55 The method of any one of claims 52-54, wherein the oligonucleotide comprising a nucleoside of formula (XI) is not linked to a solid support.
  • Embodiment 56 The method of claim 55, wherein the oligonucleotide comprising a nucleoside of formula (XI) and the oligonucleotide comprising a nucleoside of formula (X) are not linked to a solid support.
  • Embodiment 57 The method of claim 52, wherein the oligonucleotide comprising a nucleoside of formula (XI) is is linked to a solid support and the method comprises a step of cleaving the oligonucleotide from the solid support prior to reacting with the alkali hydroxide or alkali earth hydroxide (e.g., NaOH).
  • alkali hydroxide or alkali earth hydroxide e.g., NaOH
  • Embodiment 58 The method of claim 57, wherein said step of cleaving the oligonucleotide comprising a nucleoside of formula (XI) from the solid support comprises contacting the oligonucleotide linked to a solid support with an ammonium hydroxide at a temperature less than 60 °C (e.g., about room temperature or between about 20°C and 40 °C).
  • Embodiment 59 The method of any one of claims 52-58, wherein R H is fluoro.
  • Embodiment 60 The method of any one of claims 52-59, wherein at least one L is a bond.
  • Embodiment 61 The method of any one of claims 52-60, wherein at least one L is a linker.
  • Embodiment 62 The method of any one of claims 52-61, wherein at least one R L is selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucelsides and nucleotides, oligonucleotides, detectable labels, diagnostic agents (e.g., bitoin), fluorescent dyes, polyethylene glycols (PEGs), antibodies, antibody fragments (e.g., nanobodies).
  • diagnostic agents e.g., bitoin
  • fluorescent dyes e.g., polyethylene glycols (PEGs)
  • PEGs polyethylene glycols
  • antibodies e.g., nanobodies
  • Embodiment 63 The method of any one of claims 52-62, wherein at least one R L is a ligand selected from targeting ligands, endosomolytic ligands and PK modulating ligands.
  • Embodiment 64 The method of any one of claims 52-63, wherein R 6 and R 7 are same.
  • Embodiment 65 The method of any one of claims 52-64, wherein R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-6 alkoxy (e.g., methyl) or alkoxyalkyl (e.g. 2-methoxyethyl); or R 4 and R 2 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y- C(R 10 R 11 ) v -2’.
  • Embodiment 66 The method of any one of claims 52-65, wherein R 2 is hydrogen, hydroxyl, F, methoxy, or R 4 and R 2 taken together are 4 ’ -C(R 10 R 11 ) v - Y -2 ’ .
  • Embodiment 67 The method of any one claims 52-66, wherein R 4 and R 2 taken together are 4’-C(R 10 R 11 ) v -Y-2’.
  • Embodiment 68 The method of any one of claims 52-67, wherein R 4 is H.
  • Embodiment 69 The method of any one of claims 52-68, wherein R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, hydroxy, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), amino, alkylamino, dialkylamino, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a ligand, a linker covalently bonded to one or more ligands (e.g., N-acetylgalactosamine (GalNac)), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • alkoxyalkyl e.g., methoxyethyl
  • Embodiment 70 The method of any one of claims 52-69, wherein R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide, hydroxy, optionally substituted C 1-30 alkoxy, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • Embodiment 71 The method of any one of claims 52-70, wherein R 3 is a bond to an intemucleotide linkage to a subsequent nucleotide.
  • Embodiment 72 The method of any one of claims 52-71, wherein R 3 is hydroxyl.
  • Embodiment 73 The method of any one of claims 52-72, wherein R 5 is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxy, optionally substituted C 1-30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates and phosphate mimics.
  • VP vinylphosphonate
  • Embodiment 74 The method of any one of claims 52-73, wherein R 5 is a bond to an intemucleotide linkage to a preceding nucleotide.
  • Embodiment 75 The method of any one of claims 52-74, wherein R 5 is hydroxy, optionally substituted C 1-30 alkoxy, vinylphosphonate (VP) group, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate.
  • VP vinylphosphonate
  • Embodiment 76 The method of any one of claims 52-76, wherein the oligonucleotide comprises at least one ribonucleotide.
  • Embodiment 77 The method of any one of claims 52-77, wherein the oligonucleotide comprises at least one 2’-deoxyribonucleotide.
  • Embodiment 78 The method of any one of claims 52-78, wherein the oligonucleotide comprises at least one nucleotide with a modified or non-natural nucleobase.
  • Embodiment 79 The method of any one of claims 52-79, wherein the oligonucleotide comprises at least one nucleotide with a modified ribose sugar.
  • Embodiment 80 The method of any one of claims 52-80, wherein the oligonucleotide comprises at least one nucleotide comprising a group other than H or OH at the 2 ’-position of the ribose sugar.
  • Embodiment 81 The method of any one of claims 52-81, wherein the oligonucleotide comprises at least one nucleotide with a 2’-F ribose.
  • Embodiment 82 The method of any one of claims 52-82, wherein the oligonucleotide comprises at least one nucleotide with a 2’-OMe ribose.
  • Embodiment 83 The method of any one of claims 52-83, wherein the oligonucleotide comprises at least one nucleotide comprising a moiety other than a ribose sugar.
  • Embodiment 84 The method of any one of claims 52-84, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.
  • Embodiment 85 The method of any one of claims 52-85, wherein the oligonucleotide comprising a nucleoside of formula (XI) comprises at least one hydroxyl, phosphate or amino protecting group.
  • Embodiment 86 The method of any one of claims 52-86, wherein the oligonucleotide comprising a nucleoside of formula (XI) does not comprise a hydroxyl, phosphate or amino protecting group.
  • alkyl refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms.
  • alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes halo, amino, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl.
  • “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl.
  • Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.
  • a “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH 2 group to an NH group or an O group).
  • the term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof.
  • the heteroatom(s) is placed at any interior position of the heteroalkyl group.
  • up to two heteroatoms are consecutive, such as, by way of example,
  • alkenyl refers to an alkyl group containing at least one carbon-carbon double bond.
  • the alkenyl group can be optionally substituted with one or more “alkyl group substituents.”
  • Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-l-yl andheptadec-8,ll-dien-l-yl.
  • alkynyl refers to an alkyl group containing a carbon-carbon triple bond.
  • the alkynyl group can be optionally substituted with one or more “alkyl group substituents.”
  • exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl.
  • Useful alkynyl groups include the lower alkynyl groups.
  • cycloalkyl refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and/or alkylene.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl.
  • Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
  • Heterocyclyl refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively).
  • Cxheterocyclyl and Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system.
  • 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent.
  • exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4- morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3- dioxanyl, 1,4-dioxany land the like.
  • Aryl refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms.
  • the aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and — NRR', where R and R' are each independently hydrogen, alkyl, aryl and aralkyl.
  • Heteroaryl refers to an aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S ( e.g ., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.
  • Exemplary aryl and heteroaryls include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzisothi
  • halogen refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen radioisotope or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen-substituted moiety or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.
  • haloalkyl refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different.
  • fluoroalkyl and fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.
  • exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (C 1 -C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF 3 ), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like).
  • amino means -NH 2 .
  • alkylamino means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -NH(alkyl).
  • dialkylamino means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -N(alkyl)(alkyl).
  • alkylamino includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.”
  • arylamino means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl, and — N(aryl) 2 .
  • heteroarylamino means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example — NHheteroaryl, and — N(heteroaryl) 2 .
  • two substituents together with the nitrogen can also form a ring.
  • the compounds described herein containing amino moieties can include protected derivatives thereof.
  • Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like.
  • Exemplary alkylamino includes, but is not limited to, NH(C 1 - Cioalkyl), such as — NHCH 3 , — NHCH 2 CH 3 , — NHCH 2 CH 2 CH 3 , and — NHCH(CH 3 ) 2 .
  • Exemplary dialkylamino includes, but is not limited to, — N(C 1 -Cioalkyl) 2 , such as N(CH 3 ) 2 , — N(CH 2 CH 3 ) 2 , — N(CH 2 CH 2 CH 3 ) 2 , and— N(CH(CH3) 2 ) 2 .
  • aminoalkyl means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms ( — N — ) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl.
  • an (C 2 -C 6 ) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.
  • hydroxy and “hydroxyl” mean the radical — OH.
  • alkoxyl refers to an alkyl group, as defined above, having an oxygen radical attached thereto, and can be represented by one of -O-alkyl, -O- alkenyl, and -O-alkynyl.
  • Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein.
  • the alkoxy and aroxy groups can be substituted as described above for alkyl.
  • Exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n- propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O- hexyl, O- cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.
  • carbonyl means the radical — C(O) — . It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.
  • carboxy means the radical — C(O)O — . It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes -COOH, i.e., carboxyl group.
  • cyano means the radical — CN.
  • nitro means the radical — NO 2 .
  • heteroatom refers to an atom that is not a carbon atom.
  • heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens.
  • a “heteroatom moiety” includes a moiety where the atom by which the moiety is attached is not a carbon.
  • alkylthio and thioalkoxy refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur.
  • the “alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • alkylthio also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.
  • Arylthio refers to aryl or heteroaryl groups.
  • sulfmyl means the radical — SO — . It is noted that the sulfmyl radical can be further substituted with a variety of substituents to form different sulfmyl groups including sulfmic acids, sulfmamides, sulfmyl esters, sulfoxides, and the like.
  • sulfonyl means the radical — SO 2 — . It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO3H), sulfonamides, sulfonate esters, sulfones, and the like.
  • thiocarbonyl means the radical — C(S) — . It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.
  • thiocarbonyl refers to an alkyl-CO — group, wherein alkyl is as previously described. Exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and pabnitoyl.
  • Aroyl means an aryl-CO — group, wherein aryl is as previously described.
  • Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.
  • Arylthio refers to an aryl-S — group, wherein the aryl group is as previously described.
  • exemplary arylthio groups include phenylthio and naphthylthio.
  • Aralkyl refers to an aryl-alkyl — group, wherein aryl and alkyl are as previously described.
  • exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
  • Aralkyloxy refers to an aralkyl-O — group, wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxy group is benzyloxy.
  • Aralkylthio refers to an aralkyl-S — group, wherein the aralkyl group is as previously described.
  • An exemplary aralkylthio group is benzylthio.
  • Alkoxycarbonyl refers to an alkyl-O — CO — group.
  • exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-O — CO — group.
  • Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Alkoxycarbonyl refers to an aralkyl-O — CO — group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an H 2 N — CO — group.
  • Alkylcarbamoyl refers to a R'RN — CO — group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.
  • Dialkylcarbamoyl refers to R'RN — CO — group, wherein each of R and R' is independently alkyl as previously described.
  • “Acyloxy” refers to an acyl-O — group, wherein acyl is as previously described.
  • “Acylamino” refers to an acyl-NH — group, wherein acyl is as previously described.
  • “Aroylamino” refers to an aroyl-NH — group, wherein aroyl is as previously described.
  • substituted means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified.
  • substituted refers to a group “substituted” on a substituted group at any atom of the substituted group.
  • Suitable substituents include, without limitation, halogen, hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.
  • two substituents, together with the carbons to which they are attached to can form a ring.
  • an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different.
  • An “isocyanato” group refers to a NCO group.
  • a “thiocyanato” group refers to a CNS group.
  • An “isothiocyanato” group refers to a NCS group.
  • RNA e.g., mRNA
  • mRNA e.g., a transcript of a gene that encodes a protein
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein
  • target gene e.g., a target gene
  • RNA to be silenced is an endogenous gene, exogenous gene or a pathogen gene.
  • RNAs other than mRNA e.g., tRNAs, and viral RNAs, can also be targeted.
  • RNAi refers to the ability to silence, in a sequence specific manner, a target gene, e.g., mRNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.
  • a guide RNA e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity.
  • Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, /. Am. Chem. Soc. 109:3783-3785).
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary or 100% complementarity means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than perfect complementarity refers to the situation in which some, but not all, nucleoside units of two strands can hydrogen bond with each other.
  • “Substantial complementarity” refers to polynucleotide strands exhibiting 90% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as to be noncomplementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
  • the nontarget sequences typically differ by at least 5 nucleotides.
  • off-target and the phrase “off-target effects” refer to any instance in which an effector molecule against a given target causes an unintended affect by interacting either directly or indirectly with another target sequence, a DNA sequence or a cellular protein or other moiety.
  • an “off-target effect” may occur when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of an siRNA.
  • nucleoside means a glycosylamine comprising a nucleobase and a sugar. Nucleosides includes, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups. [00461] As used herein, the term “nucleotide” refers to a glycosomine comprising a nucleobase and a sugar having a phosphate group covalently linked to the sugar. Nucleotides may be modified with any of a variety of substituents.
  • locked nucleic acid or “LNA” or “locked nucleoside” or “locked nucleotide” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system. Locked nucleic acids are also referred to as bicyclic nucleic acids (BNA).
  • BNA bicyclic nucleic acids
  • methyleneoxy LNA alone refers to b-D-methyleneoxy LNA.
  • MOE refers to a 2'-O-methoxyethyl substituent.
  • the term “gapmer” refers to a chimeric oligomeric compound comprising a central region (a “gap”) and a region on either side of the central region (the “wings”), wherein the gap comprises at least one modification that is different from that of each wing.
  • modifications include nucleobase, monomeric linkage, and sugar modifications as well as the absence of modification (unmodified).
  • the nucleotide linkages in each of the wings are different than the nucleotide linkages in the gap.
  • each wing comprises nucleotides with high affinity modifications and the gap comprises nucleotides that do not comprise that modification.
  • nucleotides in the gap and the nucleotides in the wings all comprise high affinity modifications, but the high affinity modifications in the gap are different than the high affinity modifications in the wings.
  • the modifications in the wings are the same as one another. In certain embodiments, the modifications in the wings are different from each other.
  • nucleotides in the gap are unmodified and nucleotides in the wings are modified.
  • the modification(s) in each wing are the same.
  • the modification(s) in one wing are different from the modification(s) in the other wing.
  • oligomeric compounds are gapmers having 2'-deoxynucleotides in the gap and nucleotides with high-affinity modifications in the wing.
  • BNA refers to bridged nucleic acid, and is often referred as constrained or inaccessible RNA.
  • BNA can contain a 5-, 6- membered, or even a 7-membered bridged structure with a “fixed” C3 ’ -endo sugar puckering.
  • the bridge is typically incorporated at the 2 ’ 4 ’ -position of the ribose to afford a 2’, 4’-BNA nucleotide (e.g., LNA, or ENA).
  • BNA nucleotides include the following nucleosides:
  • LNA refers to locked nucleic acid, and is often referred as constrained or inaccessible RNA.
  • LNA is a modified RNA nucleotide.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge (e.g., a methylene bridge or an ethylene bridge) connecting the 2' hydroxyl to the 4' carbon of the same ribose sugar.
  • the bridge can “lock” the ribose in the 3'-endo North) conformation:
  • ⁇ NA refers to ethylene-bridged nucleic acid, and is often referred as constrained or inaccessible RNA.
  • the “cleavage site” herein means the backbone linkage in the target gene or the sense strand that is cleaved by the RISC mechanism by utilizing the iRNA agent.
  • the target cleavage site region comprises at least one or at least two nucleotides on both side of the cleavage site.
  • the cleavage site is the backbone linkage in the sense strand that would get cleaved if the sense strand itself was the target to be cleaved by the RNAi mechanism.
  • the cleavage site can be determined using methods known in the art, for example the 5 ’-RACE assay as detailed in Soutschek et al., Nature (2004) 432, 173-178, which is incorporated by reference in its entirety.
  • the cleavage site region for a conical double stranded RNAi agent comprising two 21 -nucleotides long strands (wherein the strands form a double stranded region of 19 consecutive base pairs having 2-nucleotide single stranded overhangs at the 3 ’-ends)
  • the cleavage site region corresponds to positions 9-12 from the 5 ’-end of the sense strand.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • a “terminal region” of a strand refers to positions 1-4, e.g., positions 1, 2, 3, and 4, counting from the nearest end of the strand.
  • a 5 ’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 5’-end of the strand.
  • a 3’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 3’-end of the strand.
  • a 5 ’-terminal region for the antisense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the antisense strand.
  • a preferred 5 ’-terminal region for the antisense strand is positions 1 , 2 and 3 counting from the 5 ’-end of the antisense strand.
  • a 3 ’-terminal region for the antisense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand.
  • a preferred 3 ’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 3’- end of the antisense strand.
  • a 5 ’-terminal region for the sense strand is positions 1, 2, 3 and 4 counting from the 5 ’-end of the sense strand.
  • a preferred 5 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 5 ’-end of the sense strand.
  • a 3 ’-terminal region for the sense strand can be positions 1, 2, 3, and 4 counting from the 3 ’-end of the strand.
  • a preferred 3 ’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 3 ’-end of the sense strand.
  • a “central region” of a strand refers to positions 5-17, e.g., positions 6- 16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5 ’-end of the strand.
  • the central region of a strand means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the strand.
  • a preferred central region for the sense strand is positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5’-end of the sense strand.
  • a more preferred central region for the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5 ’-end of the sense strand.
  • a preferred central region for the antisense strand is positions 9, 10, 11, 12, 13, 14, 15 16 and 17, counting from 5’-end of the antisense strand.
  • a more preferred central region for the antisense strand is positions 10, 11, 12, 13, 14, 15 and 16, counting from 5’- end of the antisense strand.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g. animal or a plant).
  • ex vivo refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube).
  • in vivo refers to events that occur within an organism (e.g. animal, plant, and/or microbe).
  • the term "subject" or "patient” refers to any organism to which a composition disclosed herein can be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “patient” and “subject” are used interchangeably herein.
  • a subject can be male or female.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders.
  • compounds, compositions and methods described herein can be used to with domesticated animals and/or pets.
  • the subject is human.
  • the subject is an experimental animal or animal substitute as a disease model.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice.
  • the term subject is further intended to include transgenic species.
  • the subject can be of European ancestry.
  • the subject can be of African American ancestry.
  • the subject can be of Asian ancestry.
  • parenteral administration refers to administration through injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
  • subcutaneous administration refers to administration just below the skin.
  • Intravenous administration means administration into a vein.
  • a dose refers to a specified quantity of a pharmaceutical agent provided in a single administration.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • a dosage unit refers to a form in which a pharmaceutical agent is provided.
  • a dosage unit is a vial comprising lyophilized antisense oligonucleotide.
  • a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
  • Example 1 Simple chemical approaches to introduce 2,6-diaminopurine and 2-aminoadenine conjugates into oligonucleotides
  • DAP 2,6-Diaminopurine
  • S-L2 cyanophage in 1977 by Kimos et al [1].
  • Previous studies showed that DAP can form three hydrogen bonds Watson-crick base-pairing with thymidine in DNA [2, 3] and uridine in RNA [4, 5] resulting on increasing duplex stability by approximately 1-2 deg/modification .
  • Tm duplex is decreased because of the steric hindrance between N2- nitrogen of DAP and sulfur of the pyrimidine base.
  • DAP building blocks were obtained from guanosine analogue but usually with a low yield [2, 3, 32-35].
  • halogenated purine such as the 6-chloro-2- aminopurine [13, 15, 16, 18, 19, 22, 36] or 2,6-dicholoropurine [37-39] analogues or the commercially available 2,6-diaminopurine [31].
  • these synthesis strategies still involve tedious chemical synthesis steps to introduce diamino function, placement of protecting groups and deprotection and purification. As the deprotection efficiency is not always perfect, the purification of the oligonucleotide is also an issue.
  • oligonucleotides comprising the DAP monomers residues show in FIG. 3.
  • Double-stranded RNA molecules i.e., siRNA duplex comprising the exemplary DAP monomer residues FIG. 2 and their biophysical studies are summarized in Table 3.
  • dsRNAs comprising the DAP monomer residues of FIG. 2 for RNAi studies are shown in Table 4.
  • FIG. 4A Exemplary post-synthetic conjugation process for post-synthesis conjugation with 2- F,6-NH2 nucleotides in solid support is shown in FIG. 4A and in solution phase in FIG. 5B.
  • Exemplary ligands used for post-synthetic conjugation include, but are not limited to, carbohydrates, peptides, lipids, diagnostic agents (Biotin), fluorescent dyes, PEGs, antibodies, antibody fragments (Nanobodies), folic acid, RGD-peptide and DUPA ligand.
  • Exemplary ligands with amine linker used for the post-synthetic conjugation process are shown in FIG. 6.
  • ESI-MS spectra were recorded on a Waters QTof Premier instrument using the direct flow injection mode.
  • 1 H NMR spectra were recorded at 500 and 600 MHz.
  • 13 C NMR spectra were recorded at 126 and 151 MHz.
  • 31 P NMR spectra were recorded at 202 and 243 MHz.
  • 19 F NMR spectra were recorded at 565 MHz.
  • Chemical shifts are given in ppm referenced to the solvent residual peak (DMSO-d 6 - 1H: d at 2.50 ppm and 13C d at 39.5 ppm; CDCl 3 - 1H: d at 7.26 ppm and 13C d at 77.16 ppm).
  • Coupling constants are given in Hertz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), septet (sept), broad signal (brs), or multiplet (m).
  • Oligomer synthesis Oligonucleotides were synthesized on an ABI-394 DNA/RNA synthesizer using modified synthesis cycles based on those provided with the instrument. A solution of 0.25 M 5 -( S-ethy lthi o)- 1H-tetrazole in acetonitrile was used as the activator. The phosphoramidite solutions were 0.15 M in anhydrous acetonitrile. The oxidizing reagent was 0.02 M I 2 in THF/pyridine/H 2 O.
  • the detritylation reagent was 3% dichloroacetic acid (DCA) in DCM.
  • DCA dichloroacetic acid
  • the solid support was washed with 0.1 M piperidine in acetonitrile for 10 min, then washed with anhydrous acetonitrile and dried with argon.
  • the oligonucleotide was then manually released from support and deprotected using a mixture of 30% NH 4 OH for 5 h at 60 °C.
  • Solvent was collected by filtration and the support was rinsed with deionized water (6 mL). About oligonucleotides containing 2'-OTBS protecting group were release from support and deprotected using NH 4 OH/ethanol (3:1, v/v) or 40% methylamine (0.5 mL/ ⁇ mol of solid support) for 6 h at 55 °C or 15 min at 60 °C, respectively. For all oligonucleotide, solvent was collected by filtration and the support was rinsed with DMSO (1.5 mL/ ⁇ mol of solid support) according to the method shown in FIG 10.
  • Crude oligonucleotides were purified by anion exchange glass column (10 x mm) packed with TSK Gel Super Q-5PW and using 20 mM sodium phosphate, 15% ACN as buffer A and 20mM sodium phosphate 15% ACN, 1M NaBr as buffer B. Purification gradient started from 20% to 60% B in 240 min at 60 °C. All single strands were purified to >90% IEX HPLC and >98% Cl 8 HPLC (260 nm) purity and then desalted by size exclusion chromatography using a custom packed with Sephadex G25 (GE Healthcare), eluted with sterile nuclease-free water. Some examples are shown in FIG 11 and 12.
  • Oligonucleotide concentrations were calculated based on absorbance at 260 nm and the following extinction coefficients: A/2,6-diaminoA, 13.86_M -1 cm -1 ; T/U, 7.92 M -1 cm -1 ; C, 6.57 M- 1 em -1 ; and G, 10.53_M -1 cm -1 .
  • the purity and identity of oligonucleotides were verified by analytical anion exchange chromatography and mass spectrometry, respectively.
  • Curent method In a vial, 26mg CPG were treated with 500 ⁇ L mixture of 28-30% NH 4 OH for 2 h at room temperature. The partially deprotected oligonucleotide was then filtrated from the solid support, purified by size exclusion chromatography and lyophized. 5 OD oligonucleotides, 100 ⁇ L of 0.2M ethanolic solution of amine and 10 ⁇ L DIPEA were solubilized in 100 ⁇ L dry DMSO and heated at 90 °C for 16h according to the method shown in Scheme 5. Illustrated example is shown in FIG 16.
  • Tm measurement UV melting curves were recorded using a Cary 3500 UV-Visible Spectrophotometer Multicell Peltier. The concentration of oligonucleotide was 1 mM, and samples were prepared in PBS buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 8 mM sodium phosphate dibasic, and 2 mM potassium phosphate monobasic, pH 7.4). Samples were annealed by heating to 85 °C and then slowly cooled to 10 °C. Samples were then heated to 95 °C at a gradient of 1 °C/min, and the change in UV absorbance at 260 nm was recorded. The melting temperature was calculated from the first derivative of the melting curve.
  • reaction mixture was extracted with ethyl acetate (5 x 1 L). After evaporation of the solvent, the crude product was obtained as yellow solid and purified by recrystallization from methanol (500 mL) at 60 °C to give (2R,5R)-5-(6-amino-2-fluoro-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran- 3-ol (22.7 g, 84.31 mmol, 56.83% yield) as yellow solid.
  • the reaction mixture was added H 2 O (500 mL), and extracted with EtOAc (500 mL x 3), the combined organic layers were washed with brine (500 mL), then dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue.
  • the residue was purified by prep-HPLC (column: Phenomenex Titank C18 Bulk 250 x 100mm 10u; mobile phase: [water(10mM NH 4 HCO 3 )-ACN]; B%: 50%-70%,20min) to afford 5'-DMT-2-F-ribo-purine (85.5 g, 145.5 mmol, 83.0% yield) as a yellow solid.
  • the reaction was stirred at 15 °C for 16 hrs.
  • the reaction was added to cooled sat. aq. NaHC03 (100 mL), then the reaction was adjusted to pH 7 ⁇ 8 with sat. aq. NaHCO 3 , extracted with EtOAc (100 mL x 15).
  • the combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a brown solid.
  • Reaction was kept for stirring at 22 °C and TLC was checked after 0.5 hr.
  • Reaction mixture was diluted with DCM (20 mL) and washed with 10% NaHCO 3 solution (20 x 2 mL).
  • Organic layer separated, dried over anhydrous Na 2 SO 4 , filtered and the filtrate was evaporated to dryness.
  • the crude mass obtained was purified by flash column chromatography (gradient: 20-50% EtOAc in hexane) to afford 12 (2.25 g, 89% yield) as yellowish white foam.
  • reaction mixture was stirred for 5 minutes at rt and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.18 g, 4.99 mmol, 1.11 mL) was added slowly into it. Reaction was kept for stirring at 22 °C and TLC was checked after 1 hr. Reaction mixture was diluted with DCM (20 mL) and washed with 10% NaHCO 3 solution (20 x 2 mL). Organic layer separated, dried over anhydrous Na 2 SO 4 , filtered and the filtrate was evaporated to dryness.
  • CPG 21 Added 11 (0.2 g, 297.77 ⁇ mol) and N-ethyl-N-isopropyl-propan-2-amine (153.93 mg, 1.19 mmol, 207.46 ⁇ L) into rb flask. Then added dry acetonitrile (50 mL). Stirred well to dissolve and then HBTU (118.57 mg, 312.65 ⁇ mol) to preactivated acid. Let stirr for ⁇ 5 min, then added CPG. Capped and put on mechanical shaker overnight. Next day filtered CPG, washed with ACN, then MeOH, then ACN, then diethyl ether.
  • CPG 22 Added 4-[(2R,5R)-5-(6-amino-2-fluoro-purin-9-yl)-2-[[bis(4- methoxyphenyl)-phenyl-methoxy]methyl]-4-[tert-butyl(dimethyl)silyl]oxy-tetrahydrofuran-3- yl]oxy-4-oxo-butanoic acid (0.74 g, 922.77 ⁇ mol) and N-ethyl-N-isopropyl-propan-2-amine (477.04 mg, 3.69 mmol, 642.91 ⁇ L) into rb flask. Then added dry acetonitrile (50 mL).
  • reaction mixture was stirred for 5 minutes at rt and 2- cyanoethyl-N,N-diisopropylchlorophosphoramidite (626.62 mg, 2.65 mmol, 591.15 ⁇ L) was added slowly into it. Reaction was kept for stirring at 22 °C and TLC was checked after 0.5 hr. Reaction mixture was diluted with DCM (20 mL) and washed with 10% NaHCO 3 solution (20 x 2 mL). Organic layer separated, dried over anhydrous Na 2 SO 4 , filtered and the filtrate was evaporated to dryness.
  • CPG 29 Added 4-[(2R,5R)-2-(6-amino-2-fluoro-purin-9-yl)-5-[[bis(4- methoxyphenyl)-phenyl-methoxy]methyl]-4-[tert-butyl(dimethyl)silyl]oxy-tetrahydrofuran-3- yl]oxy-4-oxo-butanoic acid (0.58 g, 723.26 ⁇ mol) and N-ethyl-N-isopropyl-propan-2-amine (373.89 mg, 2.89 mmol, 503.90 ⁇ L) into rb flask. Then added dry acetonitrile (50 mL).
  • Example 2 15 N labeling, 2-F A retention and Iso-G conversion in oligonucleotides derived from 2-F A building block containing oligonucleotides SCHEME 5
  • Oligonucleotide synthesis Oligonucleotides were synthesized on an ABI-394 DNA/RNA synthesizer using modified synthesis cycles based on those provided with the instrument. A solution of 0.25 M 5-(S-ethylthio)-1H-tetrazole in acetonitrile was used as the activator. The phosphoramidite solutions were 0.15 M in anhydrous acetonitrile. The oxidizing reagent was 0.02 M I2 in THF/pyridineAHO. The detritylation reagent was 3% dichloroacetic acid (DCA) in DCM. After completion of the automated synthesis, the solid support was dried with argon.
  • DCA dichloroacetic acid
  • oligonucleotide was then manually released from support and deprotected using a mixture of 30% NH 4 OH for 24h at 65 °C.
  • oligonucleotide was then manually released from support and deprotected using a mixture of 16N 15 NH 4 OH for 48h at 65 °C.
  • oligonucleotide was then manually released from support and deprotected using a mixture of 30% NH 4 OH for 18h at room temperature.
  • Oligonucleotide concentrations were calculated based on absorbance at 260 nm and the following extinction coefficients: A/2,6-diaminoA, 13.86_M -1 cm -1 ; T/U, 7.92 M -1 cm -1 ; C, 6.57 M- 1 em -1 ; and G, 10.53 M -1 cm -1 .
  • the purity and identity of oligonucleotides were verified by analytical anion exchange chromatography and mass spectrometry. Results are shown in FIGS. 18-20.

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EP22842917.1A 2021-07-15 2022-07-15 Einfache chemische ansätze zum einbringen von 2,6-diaminopurin- und 2-aminoadeninkonjugaten in oligonukleotide Pending EP4370136A2 (de)

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